LOCUS       sequence-373575-        3213 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway entry vector for sgRNA targeted to MpIGPD.Transient 
            expression of sgRNA targeted to MpIGPD in Marchantia polymorpha..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3213)
  AUTHORS   Fukushima T, Kodama Y
  TITLE     Selection of a histidine auxotrophic Marchantia polymorpha strain 
            with an auxotrophic selective marker.
  JOURNAL   Plant Biotechnol (Tokyo). 2022 Dec 25;39(4):345-354. doi: 
            10.5511/plantbiotechnology.22.0810a. Epub 2022 Nov 26.
  PUBMED    37283617
REFERENCE   2  (bases 1 to 3213)
  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"; doi: 
            "10.5511/plantbiotechnology.22.0810a"; journalName: "Plant 
            Biotechnol (Tokyo)"; date: "2022-12-25- 25"; volume: "39"; issue: 
            "4"; pages: "345-354"
COMMENT     Sequence Label: pMpGE_En03-gR085
FEATURES             Location/Qualifiers
     source          1..3213
                     /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        1221..1296
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(1338..1437)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1454..1473)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1455..1473)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1478..1494)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1478..1494)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1596..1615)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1607..2416
                     /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(1674..1693)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2563..3151
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3052..3071
                     /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 cctgctgcga tcacacaatg gttttagagc tagaaatagc aagttaaaat aaggctagtc
     1261 cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tttctagtat cgggaattct
     1321 aaagggtggg cgcgccgacc cagctttctt gtacaaagtt ggcattataa gaaagcattg
     1381 cttatcaatt tgttgcaacg aacaggtcac tatcagtcaa aataaaatca ttatttgcca
     1441 tccagctgat atcccctata gtgagtcgta ttacatggtc atagctgttt cctggcagct
     1501 ctggcccgtg tctcaaaatc tctgatgtta cattgcacaa gataaaaata tatcatcatg
     1561 aacaataaaa ctgtctgctt acataaacag taatacaagg ggtgttatga gccatattca
     1621 acgggaaacg tcgaggccgc gattaaattc caacatggat gctgatttat atgggtataa
     1681 atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc tatcgcttgt atgggaagcc
     1741 cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc gttgccaatg atgttacaga
     1801 tgagatggtc agactaaact ggctgacgga atttatgcct cttccgacca tcaagcattt
     1861 tatccgtact cctgatgatg catggttact caccactgcg atccccggaa aaacagcatt
     1921 ccaggtatta gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt
     1981 cctgcgccgg ttgcattcga ttcctgtttg taattgtcct tttaacagcg atcgcgtatt
     2041 tcgtctcgct caggcgcaat cacgaatgaa taacggtttg gttgatgcga gtgattttga
     2101 tgacgagcgt aatggctggc ctgttgaaca agtctggaaa gaaatgcata aacttttgcc
     2161 attctcaccg gattcagtcg tcactcatgg tgatttctca cttgataacc ttatttttga
     2221 cgaggggaaa ttaataggtt gtattgatgt tggacgagtc ggaatcgcag accgatacca
     2281 ggatcttgcc atcctatgga actgcctcgg tgagttttct ccttcattac agaaacggct
     2341 ttttcaaaaa tatggtattg ataatcctga tatgaataaa ttgcagtttc atttgatgct
     2401 cgatgagttt ttctaatcag aattggttaa ttggttgtaa cactggcaga gcattacgct
     2461 gacttgacgg gacggcgcaa gctcatgacc aaaatccctt aacgtgagtt acgcgtcgtt
     2521 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     2581 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     2641 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     2701 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     2761 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     2821 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     2881 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     2941 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     3001 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     3061 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     3121 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     3181 cctggccttt tgctggcctt ttgctcacat gtt
//
