LOCUS Exported 3390 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Gateway multi-site entry clone for second position mEGFP (N-terminal tag). Introduced into the genome via homology-directed repair after an LR reaction with homology sequences.. ACCESSION . VERSION . KEYWORDS pENTR-R4R3-mEGFP-N SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 3390) 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 3390) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..3390 /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..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" primer_bind complement(780..801) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1041..1060) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 1388..1409 /label=EGFP-C /note="EGFP, forward primer" protein_bind 1453..1576 /gene="mutant version of attR" /label=attR3 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" primer_bind complement(1631..1650) /label=T7 /note="T7 promoter, forward primer" promoter complement(1632..1650) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(1655..1671) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(1655..1671) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(1773..1792) /label=pENTR-R /note="pENTR vectors, reverse primer" CDS 1784..2593 /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(1851..1870) /label=Kan-R /note="Kanamycin resistance gene, reverse primer" rep_origin 2740..3328 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3229..3248 /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 gacaactttg tataataaag ttgaacgaga aacgtaaaat gatataaata 1501 tcaatatatt aaattagatt ttgcataaaa aacagactac ataatactgt aaaacacaac 1561 atatccagtc actatgaatc aactacttag atggtattag tgacctgtac tgcagggcgg 1621 ccgcgatatc ccctatagtg agtcgtatta catggtcata gctgtttcct ggcagctctg 1681 gcccgtgtct caaaatctct gatgttacat tgcacaagat aaaaatatat catcatgaac 1741 aataaaactg tctgcttaca taaacagtaa tacaaggggt gttatgagcc atattcaacg 1801 ggaaacgtcg aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg 1861 ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgcttgtatg ggaagcccga 1921 tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga 1981 gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat 2041 ccgtactcct gatgatgcat ggttactcac cactgcgatc cccggaaaaa cagcattcca 2101 ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct 2161 gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg 2221 tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga 2281 cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt 2341 ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga 2401 ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga 2461 tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt 2521 tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga 2581 tgagtttttc taatcagaat tggttaattg gttgtaacac tggcagagca ttacgctgac 2641 ttgacgggac ggcgcaagct catgaccaaa atcccttaac gtgagttacg cgtcgttcca 2701 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 2761 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 2821 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 2881 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 2941 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 3001 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 3061 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 3121 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 3181 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 3241 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 3301 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 3361 ggccttttgc tggccttttg ctcacatgtt //