LOCUS Exported 4839 bp ds-DNA circular SYN 13-MAY-2021 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 pENTR-R4R3-3xmEGFP-C 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 May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4839 /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" CDS 1458..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 2181..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 //