LOCUS Exported 4341 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION pMVP L3-L2 entry plasmid, contains Hygro-P2A + WPRE for 3- or 4-component MultiSite Gateway Pro assembly. Allows expression of C-term Hygro selection marker linked by P2A to gene in lentivirus vectors. ACCESSION . VERSION . KEYWORDS LA301: pMVP (L3-L2) P2A-Hygro + WPRE SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4341) 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 4341) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4341 /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 680..736 /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 743..1780 /codon_start=1 /product="hygromycin B phosphotransferase" /label=HygR /note="confers resistance to hygromycin" /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK EVGRV" RBS 1759..1767 /label=Shine-Dalgarno sequence /note="full consensus sequence for ribosome-binding sites upstream of start codons in E. coli; complementary to a region in the 3' end of the 16S rRNA (Chen et al., 1994)" misc_feature 1837..2425 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(1890..1910) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(2308..2319) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" protein_bind complement(2453..2552) /gene="mutant version of attL" /label=attL2 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" primer_bind complement(2582..2601) /label=T7 /note="T7 promoter, forward primer" promoter complement(2583..2601) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(2606..2622) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2606..2622) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(2724..2743) /label=pENTR-R /note="pENTR vectors, reverse primer" CDS 2735..3544 /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(2802..2821) /label=Kan-R /note="Kanamycin resistance gene, reverse primer" rep_origin 3691..4279 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4180..4199 /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 agttggtggc ggatccggag ctactaattt ctctctgtta aagcaggcag gagatgtgga 721 ggagaatcca ggtcctaagg gaatgaagaa gcccgaactc accgctacca gcgttgaaaa 781 atttctcatc gagaagttcg acagtgtgag cgacctgatg cagttgtcgg agggcgaaga 841 gagccgagcc ttcagcttcg atgtcggcgg acgcggctat gtactgcggg tgaatagctg 901 cgctgatggc ttctacaaag accgctacgt gtaccgccac ttcgccagcg ctgcactacc 961 catccccgaa gtgttggaca tcggcgagtt cagcgagagc ctgacatact gcatcagtag 1021 acgcgcccaa ggcgttactc tccaagacct ccccgaaaca gagctgcctg ctgtgttaca 1081 gcctgtcgcc gaagctatgg atgctattgc cgccgccgac ctcagtcaaa ccagcggctt 1141 cggcccattc gggccccaag gcatcggcca gtacacaacc tggcgggatt tcatttgcgc 1201 cattgctgat ccccatgtct accactggca gaccgtgatg gacgacaccg tgtccgccag 1261 cgtagctcaa gccctggacg aactgatgct gtgggccgaa gactgtcccg aggtgcgcca 1321 cctcgtccat gccgacttcg gcagcaacaa cgtcctgacc gacaacggcc gcatcaccgc 1381 cgtaatcgac tggtccgaag ctatgttcgg ggacagtcag tacgaggtgg ccaacatctt 1441 cttctggcgg ccctggctgg cttgcatgga gcagcagact cgctacttcg agcgccggca 1501 tcccgagctg gccggcagcc ctcgtctgcg agcctacatg ctgcgcatcg gcctggatca 1561 gctctaccag agcctcgtgg acggcaactt cgacgatgct gcctgggctc aaggccgctg 1621 cgatgccatc gtccgcagcg gggccggcac cgtcggtcgc acacaaatcg ctcgccggag 1681 cgcagccgta tggaccgacg gctgcgtcga ggtgctggcc gacagcggca accgccggcc 1741 cagtacacga ccgcgcgcta aggaggtagg tcgagtttaa gaattctgaa gcttagccta 1801 gagctcctat agtgagaccg gttagtaatg atcgacaatc aacctctgga ttacaaaatt 1861 tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct 1921 gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg 1981 tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc 2041 gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt 2101 cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc 2161 gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg 2221 ttgtcgggga agctgacgtc ctttccatgg ctgctcgcct gtgttgccac ctggattctg 2281 cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc 2341 ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg 2401 atctcccttt gggccgcctc cccgcctggc gatggtactg tcctagtacc tgacccagct 2461 ttcttgtaca aagttggcat tataagaaag cattgcttat caatttgttg caacgaacag 2521 gtcactatca gtcaaaataa aatcattatt tgccatccag ctgcagggcg gccgcgatat 2581 cccctatagt gagtcgtatt acatggtcat agctgtttcc tggcagctct ggcccgtgtc 2641 tcaaaatctc tgatgttaca ttgcacaaga taaaaatata tcatcatgaa caataaaact 2701 gtctgcttac ataaacagta atacaagggg tgttatgagc catattcaac gggaaacgtc 2761 gaggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat gggctcgcga 2821 taatgtcggg caatcaggtg cgacaatcta tcgcttgtat gggaagcccg atgcgccaga 2881 gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg agatggtcag 2941 actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta tccgtactcc 3001 tgatgatgca tggttactca ccactgcgat ccccggaaaa acagcattcc aggtattaga 3061 agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt 3121 gcattcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc gtctcgctca 3181 ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg acgagcgtaa 3241 tggctggcct gttgaacaag tctggaaaga aatgcataaa cttttgccat tctcaccgga 3301 ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg aggggaaatt 3361 aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg atcttgccat 3421 cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt ttcaaaaata 3481 tggtattgat aatcctgata tgaataaatt gcagtttcat ttgatgctcg atgagttttt 3541 ctaatcagaa ttggttaatt ggttgtaaca ctggcagagc attacgctga cttgacggga 3601 cggcgcaagc tcatgaccaa aatcccttaa cgtgagttac gcgtcgttcc actgagcgtc 3661 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg 3721 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct 3781 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct 3841 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct 3901 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg 3961 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc 4021 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga 4081 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg 4141 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta 4201 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg 4261 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg 4321 ctggcctttt gctcacatgt t //