LOCUS       sequence-214659-        4104 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway multi-site entry clone for second position 2x mRuby 
            (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 4104)
  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 4104)
  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-R4R3-2xmRuby-C
FEATURES             Location/Qualifiers
     source          1..4104
                     /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"
     protein_bind    2167..2290
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2345..2364)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2346..2364)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2369..2385)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2369..2385)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2487..2506)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2498..3307
                     /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(2565..2584)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3454..4042
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3943..3962
                     /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 agtaaacaac tttgtataat aaagttgaac gagaaacgta aaatgatata aatatcaata
     2221 tattaaatta gattttgcat aaaaaacaga ctacataata ctgtaaaaca caacatatcc
     2281 agtcactatg aatcaactac ttagatggta ttagtgacct gtactgcagg gcggccgcga
     2341 tatcccctat agtgagtcgt attacatggt catagctgtt tcctggcagc tctggcccgt
     2401 gtctcaaaat ctctgatgtt acattgcaca agataaaaat atatcatcat gaacaataaa
     2461 actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc aacgggaaac
     2521 gtcgaggccg cgattaaatt ccaacatgga tgctgattta tatgggtata aatgggctcg
     2581 cgataatgtc gggcaatcag gtgcgacaat ctatcgcttg tatgggaagc ccgatgcgcc
     2641 agagttgttt ctgaaacatg gcaaaggtag cgttgccaat gatgttacag atgagatggt
     2701 cagactaaac tggctgacgg aatttatgcc tcttccgacc atcaagcatt ttatccgtac
     2761 tcctgatgat gcatggttac tcaccactgc gatccccgga aaaacagcat tccaggtatt
     2821 agaagaatat cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg
     2881 gttgcattcg attcctgttt gtaattgtcc ttttaacagc gatcgcgtat ttcgtctcgc
     2941 tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg agtgattttg atgacgagcg
     3001 taatggctgg cctgttgaac aagtctggaa agaaatgcat aaacttttgc cattctcacc
     3061 ggattcagtc gtcactcatg gtgatttctc acttgataac cttatttttg acgaggggaa
     3121 attaataggt tgtattgatg ttggacgagt cggaatcgca gaccgatacc aggatcttgc
     3181 catcctatgg aactgcctcg gtgagttttc tccttcatta cagaaacggc tttttcaaaa
     3241 atatggtatt gataatcctg atatgaataa attgcagttt catttgatgc tcgatgagtt
     3301 tttctaatca gaattggtta attggttgta acactggcag agcattacgc tgacttgacg
     3361 ggacggcgca agctcatgac caaaatccct taacgtgagt tacgcgtcgt tccactgagc
     3421 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     3481 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     3541 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     3601 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     3661 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     3721 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     3781 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     3841 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     3901 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     3961 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     4021 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     4081 ttgctggcct tttgctcaca tgtt
//
