LOCUS Exported 5491 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION pMVP L3-L2 entry plasmid, contains HA tag-polyA + CMV-eGFP-P2A-TETa-polyA for 3- or 4-component MultiSite Gateway Pro assembly. Adds C-term HA tag to gene plus downstream CMV-driven GFP-P2A-TETa. ACCESSION . VERSION . KEYWORDS KA801: pMVP (L3-L2) HA tag + pA; CMV::eGFP-P2A-TETa-pA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5491) 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 5491) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5491 /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 686..712 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" polyA_signal 845..926 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" enhancer 990..1293 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 1294..1497 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 1448..1468 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" CDS 1563..2279 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS 1563..2279 /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(1608..1629) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1869..1888) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 2216..2237 /label=EGFP-C /note="EGFP, forward primer" CDS 2295..2351 /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 2361..3107 /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 3130..3251 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3141..3160) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" protein_bind complement(3603..3702) /gene="mutant version of attL" /label=attL2 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" primer_bind complement(3732..3751) /label=T7 /note="T7 promoter, forward primer" promoter complement(3733..3751) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(3756..3772) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3756..3772) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(3874..3893) /label=pENTR-R /note="pENTR vectors, reverse primer" CDS 3885..4694 /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(3952..3971) /label=Kan-R /note="Kanamycin resistance gene, reverse primer" rep_origin 4841..5429 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5330..5349 /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 aacaaagctg gatcctaccc atacgatgtg ccagattacg cttgatttaa 721 atagataact gactagagga tcataatcag ccataccaca tttgtagagg ttttacttgc 781 tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt 841 tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt 901 cacaaataaa gcattttttt cactgcctcg agtctagaat agtaatcaat tacggggtca 961 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct 1021 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta 1081 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac 1141 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt 1201 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag 1261 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat 1321 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat 1381 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc 1441 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctggt 1501 ttagtgaacc gtcagatccg ctagcgctac cggactcaga tctcgaattc gactggcgcg 1561 ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg 1621 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct 1681 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca 1741 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga 1801 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct 1861 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc 1921 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc 1981 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga 2041 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg 2101 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc 2161 actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg 2221 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagc 2281 gcgccggatc aggagctact aatttctctc tgttaaagca ggcaggagat gtggaggaga 2341 atccaggtcc tcaattcacc atgtccagac tggacaagag caaagtcata aacggcgctc 2401 tggaattact caatggagtc ggtatcgaag gcctgacgac aaggaaactc gctcaaaagc 2461 tgggagttga gcagcctacc ctgtactggc acgtgaagaa caagcgggcc ctgctcgatg 2521 ccctgccaat cgagatgctg gacaggcatc atacccactt ctgccccctg gaaggcgagt 2581 catggcaaga ctttctgcgg aacaacgcca agtcattccg ctgtgctctc ctctcacatc 2641 gcgacggggc taaagtgcat ctcggcaccc gcccaacaga gaaacagtac gaaaccctgg 2701 aaaatcagct cgcgttcctg tgtcagcaag gcttctccct ggagaacgca ctgtacgctc 2761 tgtccgccgt gggccacttt acactgggct gcgtattgga ggaacaggag catcaagtag 2821 caaaagagga aagagagaca cctaccaccg attctatgcc cccacttctg agacaagcaa 2881 ttgagctgtt cgaccggcag ggagccgaac ctgccttcct tttcggcctg gaactaatca 2941 tatgtggcct ggagaaacag ctaaagtgcg aaagcggcgg gccggccgac gcccttgacg 3001 attttgactt agacatgctc ccagccgatg cccttgacga ctttgacctt gatatgctgc 3061 ctgctgacgc tcttgacgat tttgaccttg acatgctccc cgggtaacta agtaagaatc 3121 cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa 3181 aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca 3241 ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt 3301 gggaggtttt ttaaagcaag taaaacctct acaaatgtgg tatggctgat tatgatcctg 3361 caagcctcgt cgtcctggcc ggaccacgct atctgtgcaa ggtccccggc cccggacgcg 3421 cgctccatga gcagagcgcc cgccgccgag gcgaagactc gggcggcgcc ctgcccgtcc 3481 caccaggtca acaggcggta accggcctct tcatcgggaa tgcgcgcgac cttcagcatc 3541 gccggcatgt ccccctggcg gacgggaagt atccagctcg accaggcttg tcgactcgaa 3601 gaacccagct ttcttgtaca aagttggcat tataagaaag cattgcttat caatttgttg 3661 caacgaacag gtcactatca gtcaaaataa aatcattatt tgccatccag ctgcagggcg 3721 gccgcgatat cccctatagt gagtcgtatt acatggtcat agctgtttcc tggcagctct 3781 ggcccgtgtc tcaaaatctc tgatgttaca ttgcacaaga taaaaatata tcatcatgaa 3841 caataaaact gtctgcttac ataaacagta atacaagggg tgttatgagc catattcaac 3901 gggaaacgtc gaggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat 3961 gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgcttgtat gggaagcccg 4021 atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg 4081 agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta 4141 tccgtactcc tgatgatgca tggttactca ccactgcgat ccccggaaaa acagcattcc 4201 aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc 4261 tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc 4321 gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg 4381 acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa cttttgccat 4441 tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg 4501 aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg 4561 atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt 4621 ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat ttgatgctcg 4681 atgagttttt ctaatcagaa ttggttaatt ggttgtaaca ctggcagagc attacgctga 4741 cttgacggga cggcgcaagc tcatgaccaa aatcccttaa cgtgagttac gcgtcgttcc 4801 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc 4861 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg 4921 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa 4981 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc 5041 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt 5101 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa 5161 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc 5221 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc 5281 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct 5341 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat 5401 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc 5461 tggccttttg ctggcctttt gctcacatgt t //