LOCUS Exported 4821 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Gateway multi-site entry clone for second position 3x mRuby (C-terminal tag). Introduced into the genome via homology-directed repair after an LR reaction with homology sequences.. ACCESSION . VERSION . KEYWORDS pENTR-R4R3-3xmRuby-C SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4821) 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 4821) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4821 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 92..109 /label=L4440 /note="L4440 vector, forward primer" terminator 268..295 /label=rrnB T2 terminator /note="transcription terminator T2 from the E. coli rrnB gene" terminator 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..727) /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 2884..3007 /gene="mutant version of attR" /label=attR3 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" primer_bind complement(3062..3081) /label=T7 /note="T7 promoter, forward primer" promoter complement(3063..3081) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(3086..3102) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3086..3102) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(3204..3223) /label=pENTR-R /note="pENTR vectors, reverse primer" CDS 3215..4024 /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(3282..3301) /label=Kan-R /note="Kanamycin resistance gene, reverse primer" rep_origin 4171..4759 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4660..4679 /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 ctgtacaagt 2881 aaacaacttt gtataataaa gttgaacgag aaacgtaaaa tgatataaat atcaatatat 2941 taaattagat tttgcataaa aaacagacta cataatactg taaaacacaa catatccagt 3001 cactatgaat caactactta gatggtatta gtgacctgta ctgcagggcg gccgcgatat 3061 cccctatagt gagtcgtatt acatggtcat agctgtttcc tggcagctct ggcccgtgtc 3121 tcaaaatctc tgatgttaca ttgcacaaga taaaaatata tcatcatgaa caataaaact 3181 gtctgcttac ataaacagta atacaagggg tgttatgagc catattcaac gggaaacgtc 3241 gaggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat gggctcgcga 3301 taatgtcggg caatcaggtg cgacaatcta tcgcttgtat gggaagcccg atgcgccaga 3361 gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg agatggtcag 3421 actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta tccgtactcc 3481 tgatgatgca tggttactca ccactgcgat ccccggaaaa acagcattcc aggtattaga 3541 agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt 3601 gcattcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc gtctcgctca 3661 ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg acgagcgtaa 3721 tggctggcct gttgaacaag tctggaaaga aatgcataaa cttttgccat tctcaccgga 3781 ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg aggggaaatt 3841 aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg atcttgccat 3901 cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt ttcaaaaata 3961 tggtattgat aatcctgata tgaataaatt gcagtttcat ttgatgctcg atgagttttt 4021 ctaatcagaa ttggttaatt ggttgtaaca ctggcagagc attacgctga cttgacggga 4081 cggcgcaagc tcatgaccaa aatcccttaa cgtgagttac gcgtcgttcc actgagcgtc 4141 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg 4201 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct 4261 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct 4321 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct 4381 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg 4441 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc 4501 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga 4561 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg 4621 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta 4681 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg 4741 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg 4801 ctggcctttt gctcacatgt t //