LOCUS       sequence-448010-        3442 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To make Gateway entry clone of CRISPR guide RNA under AtU6p.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3442)
  TITLE     Siddiqi August 2024 Plasmids
REFERENCE   2  (bases 1 to 3442)
  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"
COMMENT     Sequence Label: pENTR_AtU6p_BsaI_gRNA
FEATURES             Location/Qualifiers
     source          1..3442
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    569..668
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     misc_RNA        1105..1180
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(1567..1666)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1683..1702)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1684..1702)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1707..1723)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1707..1723)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1825..1844)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1836..2645
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1903..1922)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2792..3380
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3281..3300
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac
      601 ctgttcgttg caacaaattg atgagcaatg cttttttata atgccaactt tgtacaaaaa
      661 agcaggctcc gcggccgcct ttttttcttc ttcttcgttc atacagtttt tttttgttta
      721 tcagcttaca ttttcttgaa ccgtagcttt cgttttcttc tttttaactt tccattcgga
      781 gtttttgtat cttgtttcat agtttgtccc aggattagaa tgattaggca tcgaaccttc
      841 aagaatttga ttgaataaaa catcttcatt cttaagatat gaagataatc ttcaaaaggc
      901 ccctgggaat ctgaaagaag agaagcaggc ccatttatat gggaaagaac aatagtattt
      961 cttatatagg cccatttaag ttgaaaacaa tcttcaaaag tcccacatcg cttagataag
     1021 aaaacgaagc tgagtttata tacagctaga gtcgaagtag tgattgagag accaagcttc
     1081 tcgagggtac cgagctcggt ctcagtttta gagctagaaa tagcaagtta aaataaggct
     1141 agtccgttat caacttgaaa aagtggcacc gagtcggtgc tttttttggc aaaaattttc
     1201 agattttttc ttcatctgta gatttctggg tttttttttc cgtttcgtga atcataagtg
     1261 aagttttgga tgcaaatctg cgcgaaaaaa gttggacctg caatgagctt atttagatag
     1321 ctaagacaaa gtgattggtc cgttgtttca gttctgattg tcagagagtt tgtttcgagt
     1381 cggcgacacc aatgcgtttt gttaaccaga tttcgggtaa gaaatgtatc gagagtttgt
     1441 ttccagacgg ctacatcatt ttcttatgaa gggtgaaatt agatagacca aagattgaaa
     1501 cacaacattt ctttcacaaa aatataataa acttgatagc atttaggatc agccatgggc
     1561 gcgccgaccc agctttcttg tacaaagttg gcattataag aaagcattgc ttatcaattt
     1621 gttgcaacga acaggtcact atcagtcaaa ataaaatcat tatttgccat ccagctgata
     1681 tcccctatag tgagtcgtat tacatggtca tagctgtttc ctggcagctc tggcccgtgt
     1741 ctcaaaatct ctgatgttac attgcacaag ataaaaatat atcatcatga acaataaaac
     1801 tgtctgctta cataaacagt aatacaaggg gtgttatgag ccatattcaa cgggaaacgt
     1861 cgaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg
     1921 ataatgtcgg gcaatcaggt gcgacaatct atcgcttgta tgggaagccc gatgcgccag
     1981 agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca
     2041 gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc
     2101 ctgatgatgc atggttactc accactgcga tccccggaaa aacagcattc caggtattag
     2161 aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt
     2221 tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc
     2281 aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta
     2341 atggctggcc tgttgaacaa gtctggaaag aaatgcataa acttttgcca ttctcaccgg
     2401 attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat
     2461 taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca
     2521 tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat
     2581 atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt
     2641 tctaatcaga attggttaat tggttgtaac actggcagag cattacgctg acttgacggg
     2701 acggcgcaag ctcatgacca aaatccctta acgtgagtta cgcgtcgttc cactgagcgt
     2761 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     2821 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     2881 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     2941 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     3001 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     3061 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     3121 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     3181 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     3241 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     3301 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     3361 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     3421 gctggccttt tgctcacatg tt
//
