LOCUS Exported 4193 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION pMVP L3-L2 entry plasmid, contains OLLAS epitope tag + IRES2-eGFP + polyA for 3- or 4-component MultiSite Gateway Pro assembly. Allows C-term epitope fusion and IRES expression of eGFP reporter. ACCESSION . VERSION . KEYWORDS IX702: pMVP (L3-L2) OLLAS-IRES-eGFP + polyA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4193) AUTHORS Haldeman JM, Conway AE, Arlotto ME, Slentz DH, Muoio DM, Becker TC, Newgard CB TITLE Creation of versatile cloning platforms for transgene expression and dCas9-based epigenome editing. JOURNAL Nucleic Acids Res. 2018 Dec 27. pii: 5264291. doi: 10.1093/nar/gky1286. PUBMED 30590691 REFERENCE 2 (bases 1 to 4193) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4193 /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 570..665 /gene="mutant version of attL" /label=attL3 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" CDS 677..718 /codon_start=1 /product="Escherichia coli OmpF Linker and mouse Langerin fusion sequence tag (Park et al., 2008)" /label=OLLAS tag /translation="SGFANELGPRLMGK" misc_feature 722..774 /label=MCS /note="multiple cloning site" misc_feature 776..1362 /label=IRES2 /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(943..960) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 1170..1189 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 1363..2082 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS 1363..2079 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(1408..1429) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1669..1688) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 2016..2037 /label=EGFP-C /note="EGFP, forward primer" polyA_signal 2205..2286 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2242..2261) /label=SV40pA-R /note="SV40 polyA, reverse primer" protein_bind complement(2305..2404) /gene="mutant version of attL" /label=attL2 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" primer_bind complement(2434..2453) /label=T7 /note="T7 promoter, forward primer" promoter complement(2435..2453) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(2458..2474) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2458..2474) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(2576..2595) /label=pENTR-R /note="pENTR vectors, reverse primer" CDS 2587..3396 /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(2654..2673) /label=Kan-R /note="Kanamycin resistance gene, reverse primer" rep_origin 3543..4131 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4032..4051 /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 tcgggcccca aataatgatt ttattttgac tgatagtgac 601 ctgttcgttg caacaaattg atgagcaatg cttttttata atgccaactt tgtataataa 661 agttgtagct agcggaagcg gcttcgccaa cgagctggga cccaggctga tgggaaagta 721 actcgagctc aagcttcgaa ttctgcagtc gacggtaccg cgggcccggg atccgcccct 781 ctccctcccc cccccctaac gttactggcc gaagccgctt ggaataaggc cggtgtgcgt 841 ttgtctatat gttattttcc accatattgc cgtcttttgg caatgtgagg gcccggaaac 901 ctggccctgt cttcttgacg agcattccta ggggtctttc ccctctcgcc aaaggaatgc 961 aaggtctgtt gaatgtcgtg aaggaagcag ttcctctgga agcttcttga agacaaacaa 1021 cgtctgtagc gaccctttgc aggcagcgga accccccacc tggcgacagg tgcctctgcg 1081 gccaaaagcc acgtgtataa gatacacctg caaaggcggc acaaccccag tgccacgttg 1141 tgagttggat agttgtggaa agagtcaaat ggctctcctc aagcgtattc aacaaggggc 1201 tgaaggatgc ccagaaggta ccccattgta tgggatctga tctggggcct cggtacacat 1261 gctttacatg tgtttagtcg aggttaaaaa aacgtctagg ccccccgaac cacggggacg 1321 tggttttcct ttgaaaaaca cgatgataat atggccacaa ccatggtgag caagggcgag 1381 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac 1441 aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag 1501 ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc 1561 tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag 1621 tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac 1681 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg 1741 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac 1801 aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc 1861 aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac 1921 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc 1981 gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc 2041 gccgccggga tcactctcgg catggacgag ctgtacaagt aaagcggccg cgactctaga 2101 tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc 2161 tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag 2221 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt 2281 cactgcattc tagttgtggt ttgaacccag ctttcttgta caaagttggc attataagaa 2341 agcattgctt atcaatttgt tgcaacgaac aggtcactat cagtcaaaat aaaatcatta 2401 tttgccatcc agctgcaggg cggccgcgat atcccctata gtgagtcgta ttacatggtc 2461 atagctgttt cctggcagct ctggcccgtg tctcaaaatc tctgatgtta cattgcacaa 2521 gataaaaata tatcatcatg aacaataaaa ctgtctgctt acataaacag taatacaagg 2581 ggtgttatga gccatattca acgggaaacg tcgaggccgc gattaaattc caacatggat 2641 gctgatttat atgggtataa atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc 2701 tatcgcttgt atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc 2761 gttgccaatg atgttacaga tgagatggtc agactaaact ggctgacgga atttatgcct 2821 cttccgacca tcaagcattt tatccgtact cctgatgatg catggttact caccactgcg 2881 atccccggaa aaacagcatt ccaggtatta gaagaatatc ctgattcagg tgaaaatatt 2941 gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg taattgtcct 3001 tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa taacggtttg 3061 gttgatgcga gtgattttga tgacgagcgt aatggctggc ctgttgaaca agtctggaaa 3121 gaaatgcata aacttttgcc attctcaccg gattcagtcg tcactcatgg tgatttctca 3181 cttgataacc ttatttttga cgaggggaaa ttaataggtt gtattgatgt tggacgagtc 3241 ggaatcgcag accgatacca ggatcttgcc atcctatgga actgcctcgg tgagttttct 3301 ccttcattac agaaacggct ttttcaaaaa tatggtattg ataatcctga tatgaataaa 3361 ttgcagtttc atttgatgct cgatgagttt ttctaatcag aattggttaa ttggttgtaa 3421 cactggcaga gcattacgct gacttgacgg gacggcgcaa gctcatgacc aaaatccctt 3481 aacgtgagtt acgcgtcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct 3541 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta 3601 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc 3661 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac 3721 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct 3781 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat 3841 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg 3901 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa 3961 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg 4021 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga 4081 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc 4141 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gtt //