LOCUS Exported 5466 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION pMVP L3-L2 entry plasmid, contains polyA + CMV-eGFP-P2A-TETa-polyA for 3- or 4-component MultiSite Gateway Pro assembly. Adds CMV-driven GFP-P2A-TETa downstream of gene of interest.. ACCESSION . VERSION . KEYWORDS IR904: pMVP (L3-L2) pA; CMV::eGFP-P2A-TETa-pA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5466) 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 5466) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5466 /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" polyA_signal 820..901 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" enhancer 965..1268 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 1269..1472 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 1423..1443 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" CDS 1538..2254 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS 1538..2254 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(1583..1604) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1844..1863) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 2191..2212 /label=EGFP-C /note="EGFP, forward primer" CDS 2270..2326 /codon_start=1 /product="2A peptide from porcine teschovirus-1 polyprotein" /label=P2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="ATNFSLLKQAGDVEENPGP" CDS 2336..3082 /codon_start=1 /product="improved tetracycline-controlled transactivator" /label=rtTA-Advanced /note="In the Tet-On(R) system, rtTA-Advanced binds to the Tet-responsive element and stimulates transcription only in the presence of tetracycline or doxycycline." /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLDMLPA DALDDFDLDMLPADALDDFDLDMLPG" polyA_signal 3105..3226 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3116..3135) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" protein_bind complement(3578..3677) /gene="mutant version of attL" /label=attL2 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" primer_bind complement(3707..3726) /label=T7 /note="T7 promoter, forward primer" promoter complement(3708..3726) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(3731..3747) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3731..3747) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(3849..3868) /label=pENTR-R /note="pENTR vectors, reverse primer" CDS 3860..4669 /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(3927..3946) /label=Kan-R /note="Kanamycin resistance gene, reverse primer" rep_origin 4816..5404 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5305..5324 /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 agttgagatc aacaaagctt cctagctcga tttaaataga taactgacta gaggatcata 721 atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc 781 ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat 841 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg 901 cctcgagtct agaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt 961 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc 1021 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac 1081 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata 1141 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc 1201 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta 1261 ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac 1321 ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc 1381 aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc 1441 gtgtacggtg ggaggtctat ataagcagag ctggtttagt gaaccgtcag atccgctagc 1501 gctaccggac tcagatctcg aattcgactg gcgcgccatg gtgagcaagg gcgaggagct 1561 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt 1621 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat 1681 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg 1741 cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc 1801 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa 1861 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg 1921 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag 1981 ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat 2041 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc 2101 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct 2161 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc 2221 cgggatcact ctcggcatgg acgagctgta caagcgcgcc ggatcaggag ctactaattt 2281 ctctctgtta aagcaggcag gagatgtgga ggagaatcca ggtcctcaat tcaccatgtc 2341 cagactggac aagagcaaag tcataaacgg cgctctggaa ttactcaatg gagtcggtat 2401 cgaaggcctg acgacaagga aactcgctca aaagctggga gttgagcagc ctaccctgta 2461 ctggcacgtg aagaacaagc gggccctgct cgatgccctg ccaatcgaga tgctggacag 2521 gcatcatacc cacttctgcc ccctggaagg cgagtcatgg caagactttc tgcggaacaa 2581 cgccaagtca ttccgctgtg ctctcctctc acatcgcgac ggggctaaag tgcatctcgg 2641 cacccgccca acagagaaac agtacgaaac cctggaaaat cagctcgcgt tcctgtgtca 2701 gcaaggcttc tccctggaga acgcactgta cgctctgtcc gccgtgggcc actttacact 2761 gggctgcgta ttggaggaac aggagcatca agtagcaaaa gaggaaagag agacacctac 2821 caccgattct atgcccccac ttctgagaca agcaattgag ctgttcgacc ggcagggagc 2881 cgaacctgcc ttccttttcg gcctggaact aatcatatgt ggcctggaga aacagctaaa 2941 gtgcgaaagc ggcgggccgg ccgacgccct tgacgatttt gacttagaca tgctcccagc 3001 cgatgccctt gacgactttg accttgatat gctgcctgct gacgctcttg acgattttga 3061 ccttgacatg ctccccgggt aactaagtaa gaatccagac atgataagat acattgatga 3121 gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga 3181 tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg 3241 cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa 3301 cctctacaaa tgtggtatgg ctgattatga tcctgcaagc ctcgtcgtcc tggccggacc 3361 acgctatctg tgcaaggtcc ccggccccgg acgcgcgctc catgagcaga gcgcccgccg 3421 ccgaggcgaa gactcgggcg gcgccctgcc cgtcccacca ggtcaacagg cggtaaccgg 3481 cctcttcatc gggaatgcgc gcgaccttca gcatcgccgg catgtccccc tggcggacgg 3541 gaagtatcca gctcgaccag gcttgtcgac tcgaagaacc cagctttctt gtacaaagtt 3601 ggcattataa gaaagcattg cttatcaatt tgttgcaacg aacaggtcac tatcagtcaa 3661 aataaaatca ttatttgcca tccagctgca gggcggccgc gatatcccct atagtgagtc 3721 gtattacatg gtcatagctg tttcctggca gctctggccc gtgtctcaaa atctctgatg 3781 ttacattgca caagataaaa atatatcatc atgaacaata aaactgtctg cttacataaa 3841 cagtaataca aggggtgtta tgagccatat tcaacgggaa acgtcgaggc cgcgattaaa 3901 ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc 3961 aggtgcgaca atctatcgct tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca 4021 tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac 4081 ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt 4141 actcaccact gcgatccccg gaaaaacagc attccaggta ttagaagaat atcctgattc 4201 aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt cgattcctgt 4261 ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat 4321 gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga 4381 acaagtctgg aaagaaatgc ataaactttt gccattctca ccggattcag tcgtcactca 4441 tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga 4501 tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct 4561 cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc 4621 tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaat cagaattggt 4681 taattggttg taacactggc agagcattac gctgacttga cgggacggcg caagctcatg 4741 accaaaatcc cttaacgtga gttacgcgtc gttccactga gcgtcagacc ccgtagaaaa 4801 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa 4861 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc 4921 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta 4981 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct 5041 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg 5101 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag 5161 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc 5221 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg 5281 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt 5341 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg 5401 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca 5461 catgtt //