LOCUS       sequence-214657-        4818 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway multi-site entry clone for second position 3x mRuby 
            (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 4818)
  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 4818)
  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-R4R3-3xmRuby-N
FEATURES             Location/Qualifiers
     source          1..4818
                     /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             735..1445
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH
                     MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAV
                     AKFAGLGGGMDELYK"
     CDS             1452..2162
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH
                     MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAV
                     AKFAGLGGGMDELYK"
     CDS             2169..2879
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH
                     MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAV
                     AKFAGLGGGMDELYK"
     protein_bind    2881..3004
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3059..3078)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3060..3078)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3083..3099)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3083..3099)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3201..3220)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3212..4021
                     /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(3279..3298)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4168..4756
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4657..4676
                     /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 aggagctgat caaggaaaat atgcgtatga
      781 aggtggtcat ggaaggttcg gtcaacggcc accaattcaa atgcacaggt gaaggagaag
      841 gcaatccgta catgggaact caaaccatga ggatcaaagt catcgaggga ggacccctgc
      901 catttgcctt tgacattctt gccacgtcgt tcatgtatgg cagccgtact tttatcaagt
      961 acccgaaagg cattcctgat ttctttaaac agtcctttcc tgagggtttt acttgggaaa
     1021 gagttacgag atacgaagat ggtggagtcg tcaccgtcat gcaggacacc agccttgagg
     1081 atggctgtct cgtttaccac gtccaagtca gaggggtaaa ctttccctcc aatggtcccg
     1141 tgatgcagaa gaagaccaag ggttgggagc ctaatacaga gatgatgtat ccagcagatg
     1201 gtggtctgag gggatacact catatggcac tgaaagttga tggtggtggc catctgtctt
     1261 gctctttcgt aacaacttac aggtcaaaaa agaccgtcgg gaacatcaag atgcccggta
     1321 tccatgccgt tgatcaccgc ctggaaaggt tagaggaaag tgacaatgaa atgttcgtag
     1381 tacaacgcga acacgcagtt gccaagttcg ccgggcttgg tggtggcatg gacgagctgt
     1441 acaagagatc catggtgagc aagggcgagg agctgatcaa ggaaaatatg cgtatgaagg
     1501 tggtcatgga aggttcggtc aacggccacc aattcaaatg cacaggtgaa ggagaaggca
     1561 atccgtacat gggaactcaa accatgagga tcaaagtcat cgagggagga cccctgccat
     1621 ttgcctttga cattcttgcc acgtcgttca tgtatggcag ccgtactttt atcaagtacc
     1681 cgaaaggcat tcctgatttc tttaaacagt cctttcctga gggttttact tgggaaagag
     1741 ttacgagata cgaagatggt ggagtcgtca ccgtcatgca ggacaccagc cttgaggatg
     1801 gctgtctcgt ttaccacgtc caagtcagag gggtaaactt tccctccaat ggtcccgtga
     1861 tgcagaagaa gaccaagggt tgggagccta atacagagat gatgtatcca gcagatggtg
     1921 gtctgagggg atacactcat atggcactga aagttgatgg tggtggccat ctgtcttgct
     1981 ctttcgtaac aacttacagg tcaaaaaaga ccgtcgggaa catcaagatg cccggtatcc
     2041 atgccgttga tcaccgcctg gaaaggttag aggaaagtga caatgaaatg ttcgtagtac
     2101 aacgcgaaca cgcagttgcc aagttcgccg ggcttggtgg tggcatggac gagctgtaca
     2161 agagatccat ggtgagcaag ggcgaggagc tgatcaagga aaatatgcgt atgaaggtgg
     2221 tcatggaagg ttcggtcaac ggccaccaat tcaaatgcac aggtgaagga gaaggcaatc
     2281 cgtacatggg aactcaaacc atgaggatca aagtcatcga gggaggaccc ctgccatttg
     2341 cctttgacat tcttgccacg tcgttcatgt atggcagccg tacttttatc aagtacccga
     2401 aaggcattcc tgatttcttt aaacagtcct ttcctgaggg ttttacttgg gaaagagtta
     2461 cgagatacga agatggtgga gtcgtcaccg tcatgcagga caccagcctt gaggatggct
     2521 gtctcgttta ccacgtccaa gtcagagggg taaactttcc ctccaatggt cccgtgatgc
     2581 agaagaagac caagggttgg gagcctaata cagagatgat gtatccagca gatggtggtc
     2641 tgaggggata cactcatatg gcactgaaag ttgatggtgg tggccatctg tcttgctctt
     2701 tcgtaacaac ttacaggtca aaaaagaccg tcgggaacat caagatgccc ggtatccatg
     2761 ccgttgatca ccgcctggaa aggttagagg aaagtgacaa tgaaatgttc gtagtacaac
     2821 gcgaacacgc agttgccaag ttcgccgggc ttggtggtgg catggacgag ctgtacaaga
     2881 caactttgta taataaagtt gaacgagaaa cgtaaaatga tataaatatc aatatattaa
     2941 attagatttt gcataaaaaa cagactacat aatactgtaa aacacaacat atccagtcac
     3001 tatgaatcaa ctacttagat ggtattagtg acctgtactg cagggcggcc gcgatatccc
     3061 ctatagtgag tcgtattaca tggtcatagc tgtttcctgg cagctctggc ccgtgtctca
     3121 aaatctctga tgttacattg cacaagataa aaatatatca tcatgaacaa taaaactgtc
     3181 tgcttacata aacagtaata caaggggtgt tatgagccat attcaacggg aaacgtcgag
     3241 gccgcgatta aattccaaca tggatgctga tttatatggg tataaatggg ctcgcgataa
     3301 tgtcgggcaa tcaggtgcga caatctatcg cttgtatggg aagcccgatg cgccagagtt
     3361 gtttctgaaa catggcaaag gtagcgttgc caatgatgtt acagatgaga tggtcagact
     3421 aaactggctg acggaattta tgcctcttcc gaccatcaag cattttatcc gtactcctga
     3481 tgatgcatgg ttactcacca ctgcgatccc cggaaaaaca gcattccagg tattagaaga
     3541 atatcctgat tcaggtgaaa atattgttga tgcgctggca gtgttcctgc gccggttgca
     3601 ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc
     3661 gcaatcacga atgaataacg gtttggttga tgcgagtgat tttgatgacg agcgtaatgg
     3721 ctggcctgtt gaacaagtct ggaaagaaat gcataaactt ttgccattct caccggattc
     3781 agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg ggaaattaat
     3841 aggttgtatt gatgttggac gagtcggaat cgcagaccga taccaggatc ttgccatcct
     3901 atggaactgc ctcggtgagt tttctccttc attacagaaa cggctttttc aaaaatatgg
     3961 tattgataat cctgatatga ataaattgca gtttcatttg atgctcgatg agtttttcta
     4021 atcagaattg gttaattggt tgtaacactg gcagagcatt acgctgactt gacgggacgg
     4081 cgcaagctca tgaccaaaat cccttaacgt gagttacgcg tcgttccact gagcgtcaga
     4141 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     4201 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     4261 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     4321 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     4381 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     4441 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     4501 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     4561 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     4621 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     4681 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     4741 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     4801 gccttttgct cacatgtt
//
