LOCUS       sequence-220022-        3390 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway multi-site entry clone for second position mEGFP (N-terminal
            tag). Introduced into the genome via homology-directed repair after 
            an LR reaction with homology sequences..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3390)
  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 3390)
  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-R4R3-mEGFP-N
FEATURES             Location/Qualifiers
     source          1..3390
                     /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    complement(603..726)
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             join(735..737,738..740,741..1451)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(780..801)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1041..1060)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1388..1409
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    1453..1576
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1631..1650)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1632..1650)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1655..1671)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1655..1671)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1773..1792)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1784..2593
                     /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(1851..1870)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2740..3328
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3229..3248
                     /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 tcgggcccct acaggtcact aataccatct aagtagttga
      601 ttcatagtga ctggatatgt tgtgttttac agtattatgt agtctgtttt ttatgcaaaa
      661 tctaatttaa tatattgata tttatatcat tttacgtttc tcgttcaact tttctataca
      721 aagttgaagg atccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
      781 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
      841 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
      901 tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc
      961 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
     1021 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
     1081 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     1141 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg
     1201 acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca
     1261 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     1321 tgcccgacaa ccactacctg agcacccagt ccaagcttag caaagacccc aacgagaagc
     1381 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     1441 agctgtacaa gacaactttg tataataaag ttgaacgaga aacgtaaaat gatataaata
     1501 tcaatatatt aaattagatt ttgcataaaa aacagactac ataatactgt aaaacacaac
     1561 atatccagtc actatgaatc aactacttag atggtattag tgacctgtac tgcagggcgg
     1621 ccgcgatatc ccctatagtg agtcgtatta catggtcata gctgtttcct ggcagctctg
     1681 gcccgtgtct caaaatctct gatgttacat tgcacaagat aaaaatatat catcatgaac
     1741 aataaaactg tctgcttaca taaacagtaa tacaaggggt gttatgagcc atattcaacg
     1801 ggaaacgtcg aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg
     1861 ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgcttgtatg ggaagcccga
     1921 tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga
     1981 gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat
     2041 ccgtactcct gatgatgcat ggttactcac cactgcgatc cccggaaaaa cagcattcca
     2101 ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct
     2161 gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg
     2221 tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga
     2281 cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt
     2341 ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga
     2401 ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga
     2461 tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt
     2521 tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga
     2581 tgagtttttc taatcagaat tggttaattg gttgtaacac tggcagagca ttacgctgac
     2641 ttgacgggac ggcgcaagct catgaccaaa atcccttaac gtgagttacg cgtcgttcca
     2701 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     2761 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     2821 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     2881 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     2941 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     3001 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     3061 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     3121 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     3181 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     3241 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     3301 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     3361 ggccttttgc tggccttttg ctcacatgtt
//
