LOCUS       sequence-214639-        3018 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway entry vector containing attL1 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 3018)
  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 3018)
  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-L1L4
FEATURES             Location/Qualifiers
     source          1..3018
                     /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    complement(1133..1228)
                     /gene="mutant version of attL"
                     /label=attL4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1259..1278)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1260..1278)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1283..1299)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1283..1299)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1401..1420)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1412..2221
                     /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(1479..1498)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2368..2956
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2857..2876
                     /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 tcgagctcca cccaactttt
     1141 ctatacaaag ttggcattat aagaaagcat tgcttatcaa tttgttgcaa cgaacaggtc
     1201 actatcagtc aaaataaaat cattatttgc catccagctg cagggcggcc gcgatatccc
     1261 ctatagtgag tcgtattaca tggtcatagc tgtttcctgg cagctctggc ccgtgtctca
     1321 aaatctctga tgttacattg cacaagataa aaatatatca tcatgaacaa taaaactgtc
     1381 tgcttacata aacagtaata caaggggtgt tatgagccat attcaacggg aaacgtcgag
     1441 gccgcgatta aattccaaca tggatgctga tttatatggg tataaatggg ctcgcgataa
     1501 tgtcgggcaa tcaggtgcga caatctatcg cttgtatggg aagcccgatg cgccagagtt
     1561 gtttctgaaa catggcaaag gtagcgttgc caatgatgtt acagatgaga tggtcagact
     1621 aaactggctg acggaattta tgcctcttcc gaccatcaag cattttatcc gtactcctga
     1681 tgatgcatgg ttactcacca ctgcgatccc cggaaaaaca gcattccagg tattagaaga
     1741 atatcctgat tcaggtgaaa atattgttga tgcgctggca gtgttcctgc gccggttgca
     1801 ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc
     1861 gcaatcacga atgaataacg gtttggttga tgcgagtgat tttgatgacg agcgtaatgg
     1921 ctggcctgtt gaacaagtct ggaaagaaat gcataaactt ttgccattct caccggattc
     1981 agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg ggaaattaat
     2041 aggttgtatt gatgttggac gagtcggaat cgcagaccga taccaggatc ttgccatcct
     2101 atggaactgc ctcggtgagt tttctccttc attacagaaa cggctttttc aaaaatatgg
     2161 tattgataat cctgatatga ataaattgca gtttcatttg atgctcgatg agtttttcta
     2221 atcagaattg gttaattggt tgtaacactg gcagagcatt acgctgactt gacgggacgg
     2281 cgcaagctca tgaccaaaat cccttaacgt gagttacgcg tcgttccact gagcgtcaga
     2341 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     2401 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     2461 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     2521 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     2581 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     2641 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     2701 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     2761 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     2821 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     2881 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     2941 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     3001 gccttttgct cacatgtt
//
