LOCUS       sequence-238960-        4116 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway multi-site entry clone for second position 2x mEGFP 
            (C-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 4116)
  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 4116)
  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-R4R3-2xmEGFP-C
FEATURES             Location/Qualifiers
     source          1..4116
                     /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"
     CDS             join(1458..1460,1461..1463,1464..2177)
                     /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"
     protein_bind    2179..2302
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2357..2376)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2358..2376)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2381..2397)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2381..2397)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2499..2518)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2510..3319
                     /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(2577..2596)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3466..4054
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3955..3974
                     /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 gagatccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
     1501 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
     1561 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
     1621 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct
     1681 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
     1741 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
     1801 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
     1861 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg
     1921 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg
     1981 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     2041 tgctgcccga caaccactac ctgagcaccc agtccaagct tagcaaagac cccaacgaga
     2101 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
     2161 acgagctgta caagtaaaca actttgtata ataaagttga acgagaaacg taaaatgata
     2221 taaatatcaa tatattaaat tagattttgc ataaaaaaca gactacataa tactgtaaaa
     2281 cacaacatat ccagtcacta tgaatcaact acttagatgg tattagtgac ctgtactgca
     2341 gggcggccgc gatatcccct atagtgagtc gtattacatg gtcatagctg tttcctggca
     2401 gctctggccc gtgtctcaaa atctctgatg ttacattgca caagataaaa atatatcatc
     2461 atgaacaata aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat
     2521 tcaacgggaa acgtcgaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta
     2581 taaatgggct cgcgataatg tcgggcaatc aggtgcgaca atctatcgct tgtatgggaa
     2641 gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac
     2701 agatgagatg gtcagactaa actggctgac ggaatttatg cctcttccga ccatcaagca
     2761 ttttatccgt actcctgatg atgcatggtt actcaccact gcgatccccg gaaaaacagc
     2821 attccaggta ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt
     2881 gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt
     2941 atttcgtctc gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt
     3001 tgatgacgag cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataaactttt
     3061 gccattctca ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt
     3121 tgacgagggg aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata
     3181 ccaggatctt gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg
     3241 gctttttcaa aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat
     3301 gctcgatgag tttttctaat cagaattggt taattggttg taacactggc agagcattac
     3361 gctgacttga cgggacggcg caagctcatg accaaaatcc cttaacgtga gttacgcgtc
     3421 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     3481 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     3541 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     3601 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     3661 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     3721 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     3781 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     3841 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     3901 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     3961 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     4021 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     4081 gttcctggcc ttttgctggc cttttgctca catgtt
//
