LOCUS       sequence-219773-        3067 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway entry vector containing attL1 and attR5 sites, U6 promoter, 
            BsaI sites for protospacer ligation, and sgRNA..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3067)
  AUTHORS   Mallett DR, Chang M, Cheng X, Bezanilla M
  TITLE     Efficient and modular CRISPR-Cas9 vector system for Physcomitrella 
            patens.
  JOURNAL   Plant Direct. 2019 Sep 12;3(9):e00168. doi: 10.1002/pld3.168. 
            eCollection 2019 Sep.
  PUBMED    31523744
REFERENCE   2  (bases 1 to 3067)
  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 
            Direct."; date: "2019-09-12"; volume: "3(9):e00168. doi"; pages: " 
            10.1002/pld3.168. eCollection 2019 Sep"
COMMENT     Sequence Label: pENTR-PpU6P-sgRNA-L1R5
FEATURES             Location/Qualifiers
     source          1..3067
                     /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"
     protein_bind    1130..1253
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1308..1327)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1309..1327)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1332..1348)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1332..1348)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1450..1469)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1461..2270
                     /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(1528..1547)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2417..3005
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2906..2925
                     /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 ccttcaccgt cagatgctac tactaaaaaa aagcaccgac tcggtgccac
      721 tttttcaagt tgataacgga ctagccttat tttaacttgc tatgcttttc agcatagctc
      781 taaaactgag accttggtct ctatgggttg taagtcctcc accttcctat atacccgcct
      841 tcacacccac ctccattcca tgtgggttat atccctcgct ccatcccttt tctccccccc
      901 ccctcgaaac ccggtcgcgt ccactcggcc agccacccag ctggacccac tcactcactg
      961 caccctttct gcttgtgctc tcccgctgcc gctccttcgg gatatcccct tctccgccac
     1021 cgcaaggacg ccgccgtgga agaaccacac catcacatca catcacatct acacccatcg
     1081 tcgctaacct gtcgcaacac gtctgcttca atggacgaat tcgagctcac aactttgtat
     1141 acaaaagttg aacgagaaac gtaaaatgat ataaatatca atatattaaa ttagattttg
     1201 cataaaaaac agactacata atactgtaaa acacaacata tccagtcact atgaatcaac
     1261 tacttagatg gtattagtga cctgtactgc agggcggccg cgatatcccc tatagtgagt
     1321 cgtattacat ggtcatagct gtttcctggc agctctggcc cgtgtctcaa aatctctgat
     1381 gttacattgc acaagataaa aatatatcat catgaacaat aaaactgtct gcttacataa
     1441 acagtaatac aaggggtgtt atgagccata ttcaacggga aacgtcgagg ccgcgattaa
     1501 attccaacat ggatgctgat ttatatgggt ataaatgggc tcgcgataat gtcgggcaat
     1561 caggtgcgac aatctatcgc ttgtatggga agcccgatgc gccagagttg tttctgaaac
     1621 atggcaaagg tagcgttgcc aatgatgtta cagatgagat ggtcagacta aactggctga
     1681 cggaatttat gcctcttccg accatcaagc attttatccg tactcctgat gatgcatggt
     1741 tactcaccac tgcgatcccc ggaaaaacag cattccaggt attagaagaa tatcctgatt
     1801 caggtgaaaa tattgttgat gcgctggcag tgttcctgcg ccggttgcat tcgattcctg
     1861 tttgtaattg tccttttaac agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa
     1921 tgaataacgg tttggttgat gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg
     1981 aacaagtctg gaaagaaatg cataaacttt tgccattctc accggattca gtcgtcactc
     2041 atggtgattt ctcacttgat aaccttattt ttgacgaggg gaaattaata ggttgtattg
     2101 atgttggacg agtcggaatc gcagaccgat accaggatct tgccatccta tggaactgcc
     2161 tcggtgagtt ttctccttca ttacagaaac ggctttttca aaaatatggt attgataatc
     2221 ctgatatgaa taaattgcag tttcatttga tgctcgatga gtttttctaa tcagaattgg
     2281 ttaattggtt gtaacactgg cagagcatta cgctgacttg acgggacggc gcaagctcat
     2341 gaccaaaatc ccttaacgtg agttacgcgt cgttccactg agcgtcagac cccgtagaaa
     2401 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     2461 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     2521 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     2581 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     2641 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     2701 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     2761 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     2821 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     2881 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     2941 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     3001 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     3061 acatgtt
//
