LOCUS       sequence-214654-        4839 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway multi-site entry clone for second position 3x 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 4839)
  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 4839)
  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-R4R3-3xmEGFP-C
FEATURES             Location/Qualifiers
     source          1..4839
                     /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..2174)
                     /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(2181..2183,2184..2186,2187..2900)
                     /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    2902..3025
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3080..3099)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3081..3099)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3104..3120)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3104..3120)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3222..3241)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3233..4042
                     /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(3300..3319)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4189..4777
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4678..4697
                     /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 caagagatcc atggtgagca agggcgagga gctgttcacc ggggtggtgc
     2221 ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg
     2281 gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc
     2341 tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc
     2401 gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg
     2461 tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga
     2521 agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg
     2581 acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca
     2641 tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg
     2701 acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg
     2761 tgctgctgcc cgacaaccac tacctgagca cccagtccaa gcttagcaaa gaccccaacg
     2821 agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca
     2881 tggacgagct gtacaagtaa acaactttgt ataataaagt tgaacgagaa acgtaaaatg
     2941 atataaatat caatatatta aattagattt tgcataaaaa acagactaca taatactgta
     3001 aaacacaaca tatccagtca ctatgaatca actacttaga tggtattagt gacctgtact
     3061 gcagggcggc cgcgatatcc cctatagtga gtcgtattac atggtcatag ctgtttcctg
     3121 gcagctctgg cccgtgtctc aaaatctctg atgttacatt gcacaagata aaaatatatc
     3181 atcatgaaca ataaaactgt ctgcttacat aaacagtaat acaaggggtg ttatgagcca
     3241 tattcaacgg gaaacgtcga ggccgcgatt aaattccaac atggatgctg atttatatgg
     3301 gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctatc gcttgtatgg
     3361 gaagcccgat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt
     3421 tacagatgag atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa
     3481 gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc ccggaaaaac
     3541 agcattccag gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc
     3601 agtgttcctg cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg
     3661 cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga
     3721 ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataaact
     3781 tttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat
     3841 ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg
     3901 ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa
     3961 acggcttttt caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt
     4021 gatgctcgat gagtttttct aatcagaatt ggttaattgg ttgtaacact ggcagagcat
     4081 tacgctgact tgacgggacg gcgcaagctc atgaccaaaa tcccttaacg tgagttacgc
     4141 gtcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     4201 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     4261 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     4321 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     4381 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     4441 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     4501 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     4561 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     4621 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     4681 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     4741 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     4801 acggttcctg gccttttgct ggccttttgc tcacatgtt
//
