LOCUS       Exported                3214 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Used to construct a gRNA expression cassette for CRISPR/Cas9 genome 
            editing in Marchantia polymorpha.
ACCESSION   .
VERSION     .
KEYWORDS    pMpGE_En03
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3214)
  AUTHORS   Sugano SS, Nishihama R, Shirakawa M, Takagi J, Matsuda Y, Ishida S, 
            Shimada T, Hara-Nishimura I, Osakabe K, Kohchi T
  TITLE     Efficient CRISPR/Cas9-based genome editing and its application to 
            conditional genetic analysis in Marchantia polymorpha.
  JOURNAL   PLoS One. 2018 Oct 31;13(10):e0205117. doi: 
            10.1371/journal.pone.0205117. eCollection 2018.
  PUBMED    30379827
REFERENCE   2  (bases 1 to 3214)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..3214
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      268..295
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      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        1222..1297
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(1339..1438)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1455..1474)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1456..1474)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1479..1495)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1479..1495)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1597..1616)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1608..2417
                     /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(1675..1694)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2564..3152
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3053..3072
                     /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 gcggccgccc ccttgtaacg tgagactact acactcctca acgcaagttc
      721 tatctcatat ctctgcaaga gcttattcca agaaactttc ttgtgcttct atgcatgccc
      781 aggtacctta agtcgcgcgc atagtcactt cacagccttt tcagttctcc ctttgatggc
      841 tcagagtgac gcaactgcac aaaaataaga aaaaagtcga agcgtcacga gagatgtatc
      901 cggctagttt ccttggagtt cggattgctc tctttctctg tctccccgaa ccatatggaa
      961 tttatgaact gctgttcact agtcagtcac ttgtttcaga acttttgagt ggtcgattta
     1021 tccaccttct tgtttcttcc tgggtccaga atctgacttc ttgagacaac aaacagtgca
     1081 cagtggatcg aagaatgacc acagttgaga cacgtggcgc tggcctcatt tattgcacca
     1141 tacgtttagt gaagaactga ataatggtgg atataaagca gagttgcacc cagcctctcg
     1201 ggagaccgag agagggtctc agttttagag ctagaaatag caagttaaaa taaggctagt
     1261 ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt ttttctagta tcgggaattc
     1321 taaagggtgg gcgcgccgac ccagctttct tgtacaaagt tggcattata agaaagcatt
     1381 gcttatcaat ttgttgcaac gaacaggtca ctatcagtca aaataaaatc attatttgcc
     1441 atccagctga tatcccctat agtgagtcgt attacatggt catagctgtt tcctggcagc
     1501 tctggcccgt gtctcaaaat ctctgatgtt acattgcaca agataaaaat atatcatcat
     1561 gaacaataaa actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc
     1621 aacgggaaac gtcgaggccg cgattaaatt ccaacatgga tgctgattta tatgggtata
     1681 aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgcttg tatgggaagc
     1741 ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat gatgttacag
     1801 atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc atcaagcatt
     1861 ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccccgga aaaacagcat
     1921 tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt
     1981 tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc gatcgcgtat
     2041 ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg agtgattttg
     2101 atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat aaacttttgc
     2161 cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac cttatttttg
     2221 acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca gaccgatacc
     2281 aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta cagaaacggc
     2341 tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt catttgatgc
     2401 tcgatgagtt tttctaatca gaattggtta attggttgta acactggcag agcattacgc
     2461 tgacttgacg ggacggcgca agctcatgac caaaatccct taacgtgagt tacgcgtcgt
     2521 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     2581 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     2641 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     2701 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     2761 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     2821 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     2881 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     2941 acctacagcg tgagcattga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     3001 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     3061 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     3121 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     3181 tcctggcctt ttgctggcct tttgctcaca tgtt
//
