LOCUS       sequence-214644-        3015 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway entry vector containing attL5 and attL4 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 3015)
  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 3015)
  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-L5L4
FEATURES             Location/Qualifiers
     source          1..3015
                     /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    570..665
                     /gene="mutant version of attL"
                     /label=attL5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(1130..1225)
                     /gene="mutant version of attL"
                     /label=attL4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1256..1275)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1257..1275)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1280..1296)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1280..1296)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1398..1417)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1409..2218
                     /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(1476..1495)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2365..2953
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2854..2873
                     /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 tgtatacaaa
      661 agttgcccct tcaccgtcag atgctactac taaaaaaaag caccgactcg gtgccacttt
      721 ttcaagttga taacggacta gccttatttt aacttgctat gcttttcagc atagctctaa
      781 aactgagacc ttggtctcta tgggttgtaa gtcctccacc ttcctatata cccgccttca
      841 cacccacctc cattccatgt gggttatatc cctcgctcca tcccttttct cccccccccc
      901 tcgaaacccg gtcgcgtcca ctcggccagc cacccagctg gacccactca ctcactgcac
      961 cctttctgct tgtgctctcc cgctgccgct ccttcgggat atccccttct ccgccaccgc
     1021 aaggacgccg ccgtggaaga accacaccat cacatcacat cacatctaca cccatcgtcg
     1081 ctaacctgtc gcaacacgtc tgcttcaatg gacgaattcg agctccaccc aacttttcta
     1141 tacaaagttg gcattataag aaagcattgc ttatcaattt gttgcaacga acaggtcact
     1201 atcagtcaaa ataaaatcat tatttgccat ccagctgcag ggcggccgcg atatccccta
     1261 tagtgagtcg tattacatgg tcatagctgt ttcctggcag ctctggcccg tgtctcaaaa
     1321 tctctgatgt tacattgcac aagataaaaa tatatcatca tgaacaataa aactgtctgc
     1381 ttacataaac agtaatacaa ggggtgttat gagccatatt caacgggaaa cgtcgaggcc
     1441 gcgattaaat tccaacatgg atgctgattt atatgggtat aaatgggctc gcgataatgt
     1501 cgggcaatca ggtgcgacaa tctatcgctt gtatgggaag cccgatgcgc cagagttgtt
     1561 tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg tcagactaaa
     1621 ctggctgacg gaatttatgc ctcttccgac catcaagcat tttatccgta ctcctgatga
     1681 tgcatggtta ctcaccactg cgatccccgg aaaaacagca ttccaggtat tagaagaata
     1741 tcctgattca ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc
     1801 gattcctgtt tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca
     1861 atcacgaatg aataacggtt tggttgatgc gagtgatttt gatgacgagc gtaatggctg
     1921 gcctgttgaa caagtctgga aagaaatgca taaacttttg ccattctcac cggattcagt
     1981 cgtcactcat ggtgatttct cacttgataa ccttattttt gacgagggga aattaatagg
     2041 ttgtattgat gttggacgag tcggaatcgc agaccgatac caggatcttg ccatcctatg
     2101 gaactgcctc ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa aatatggtat
     2161 tgataatcct gatatgaata aattgcagtt tcatttgatg ctcgatgagt ttttctaatc
     2221 agaattggtt aattggttgt aacactggca gagcattacg ctgacttgac gggacggcgc
     2281 aagctcatga ccaaaatccc ttaacgtgag ttacgcgtcg ttccactgag cgtcagaccc
     2341 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     2401 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     2461 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     2521 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     2581 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     2641 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     2701 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     2761 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     2821 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     2881 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     2941 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     3001 ttttgctcac atgtt
//
