LOCUS Exported 8338 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION sgRNA + 2x MS2(wt+f6) with MCP-VP64 effector for mammalian cells, marked by BFP. ACCESSION . VERSION . KEYWORDS pJZC116 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8338) AUTHORS Zalatan JG, Lee ME, Almeida R, Gilbert LA, Whitehead EH, La Russa M, Tsai JC, Weissman JS, Dueber JE, Qi LS, Lim WA TITLE Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds. JOURNAL Cell. 2014 Dec 18. pii: S0092-8674(14)01570-0. doi: 10.1016/j.cell.2014.11.052. PUBMED 25533786 REFERENCE 2 (bases 1 to 8338) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8338 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(56..75) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 247..626 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 628..826 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" LTR 844..1024 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 1071..1196 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1695..1928 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 2113..2157 /codon_start=1 /product="antigenic peptide corresponding to amino acids 655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik et al., 2013)" /label=gp41 peptide /note="recognized by the 2H10 single-chain llama nanobody" /translation="KNEQELLELDKWASL" misc_feature 2455..2572 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" protein_bind 2825..2858 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind 2862..2882 /label=mU6-F /note="Mouse U6 promoter, forward primer" misc_RNA 3060..3078 /label=MS2 stem loop /note="stem loop that binds the bacteriophage MS2 coat protein" terminator 3110..3129 /label=SUP4 terminator /note="transcription terminator for the S. cerevisiae SUP4 tRNA gene" enhancer 3261..3564 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 3565..3768 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" CDS 3805..3825 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 4198..4347 /codon_start=1 /product="tetrameric repeat of the minimal activation domain of herpes simplex virus VP16 (Beerli et al., 1998)" /label=VP64 /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF DLDML" CDS 4363..4419 /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 4420..5118 /codon_start=1 /product="monomeric blue fluorescent protein" /label=TagBFP /note="mammalian codon-optimized" /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD LPSKLGHKLN" protein_bind 5138..5171 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 5227..5815 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(5280..5300) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(5698..5709) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(5818..5834) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5819..5835) /label=pBluescriptKS /note="For pBluescript vector" LTR 6344..6524 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" rep_origin complement(6586..7174) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6666..6685) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(7345..8205) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 7968..7987 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(8206..8310) /gene="bla" /label=AmpR promoter ORIGIN 1 gcttaagcgg tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa 61 tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg 121 ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca 181 tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata 241 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc 301 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac 361 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa 421 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag 481 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc 541 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct 601 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg 661 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt 721 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga 781 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagcgcg ttttgcctgt 841 actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac 901 ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg 961 ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct 1021 agcagtggcg cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac 1081 gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta 1141 cgccaaaaat tttgactagc ggaggctaga aggagagaga tgggtgcgag agcgtcagta 1201 ttaagcgggg gagaattaga tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga 1261 aaaaatataa attaaaacat atagtatggg caagcaggga gctagaacga ttcgcagtta 1321 atcctggcct gttagaaaca tcagaaggct gtagacaaat actgggacag ctacaaccat 1381 cccttcagac aggatcagaa gaacttagat cattatataa tacagtagca accctctatt 1441 gtgtgcatca aaggatagag ataaaagaca ccaaggaagc tttagacaag atagaggaag 1501 agcaaaacaa aagtaagacc accgcacagc aagcggccgg ccgcgctgat cttcagacct 1561 ggaggaggag atatgaggga caattggaga agtgaattat ataaatataa agtagtaaaa 1621 attgaaccat taggagtagc acccaccaag gcaaagagaa gagtggtgca gagagaaaaa 1681 agagcagtgg gaataggagc tttgttcctt gggttcttgg gagcagcagg aagcactatg 1741 ggcgcagcgt caatgacgct gacggtacag gccagacaat tattgtctgg tatagtgcag 1801 cagcagaaca atttgctgag ggctattgag gcgcaacagc atctgttgca actcacagtc 1861 tggggcatca agcagctcca ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa 1921 cagctcctgg ggatttgggg ttgctctgga aaactcattt gcaccactgc tgtgccttgg 1981 aatgctagtt ggagtaataa atctctggaa cagatttgga atcacacgac ctggatggag 2041 tgggacagag aaattaacaa ttacacaagc ttaatacact ccttaattga agaatcgcaa 2101 aaccagcaag aaaagaatga acaagaatta ttggaattag ataaatgggc aagtttgtgg 2161 aattggttta acataacaaa ttggctgtgg tatataaaat tattcataat gatagtagga 2221 ggcttggtag gtttaagaat agtttttgct gtactttcta tagtgaatag agttaggcag 2281 ggatattcac cattatcgtt tcagacccac ctcccaaccc cgaggggacc cgacaggccc 2341 gaaggaatag aagaagaagg tggagagaga gacagagaca gatccattcg attagtgaac 2401 ggatcggcac tgcgtgcgcc aattctgcag acaaatggca gtattcatcc acaattttaa 2461 aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac 2521 agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt ttcgggttta 2581 ttacagggac agcagagatc cagtttggtt agtaccgggc ccgctctaga gatccgacgc 2641 gccatctcta ggcccgcgcc ggccccctcg cacagacttg tgggagaagc tcggctactc 2701 ccctgccccg gttaatttgc atataatatt tcctagtaac tatagaggct taatgtgcga 2761 taaaagacag ataatctgtt ctttttaata ctagctacat tttacatgat aggcttggat 2821 ttctataact tcgtatagca tacattatac gaagttataa acagcacaaa aggaaactca 2881 ccctaactgt aaagtaattg tgtgttttga gactataagt atcccttgga gaaccacctt 2941 gttggcagac gcgaggaagg agggcgcgtt taagagctat gctggaaaca gcatagcaag 3001 tttaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcgggagca 3061 catgaggatc acccatgtgc gactcccaca gtcactgggg agtcttccct ttttttgttt 3121 tttatgtctc tgcagagttc ggtctcgagt actaggatcc attaggcggc cgcgtggata 3181 accgtattac cgccatgcat tagttattaa tagtaatcaa ttacggggtc attagttcat 3241 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg 3301 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata 3361 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta 3421 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc 3481 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac 3541 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga 3601 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg 3661 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg 3721 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctgg tttagtgaac 3781 cgtcagatcc gctagcccac catgcccaaa aagaaaagaa aagtgggtag tatggcttct 3841 aactttactc agttcgttct cgtcgacaat ggcggaactg gcgacgtgac tgtcgcccca 3901 agcaacttcg ctaacgggat cgctgaatgg atcagctcta actcgcgttc acaggcttac 3961 aaagtaacct gtagcgttcg tcagagctct gcgcagaatc gcaaatacac catcaaagtc 4021 gaggtgccta aaggcgcctg gcgttcgtac ttaaatatgg aactaaccat tccaattttc 4081 gccacgaatt ccgactgcga gcttattgtt aaggcaatgc aaggtctcct aaaagatgga 4141 aacccgattc cctcagcaat cgcagcaaac tccggcatct acggctccgg acgagccgat 4201 gctctcgacg attttgacct cgatatgctg ggatccgacg ctctcgatga cttcgacctt 4261 gacatgctgg ggagtgacgc tctggacgac ttcgatctcg acatgctggg ctccgacgcc 4321 ctggatgact tcgatctgga catgctcatc aacggcagcg gcgctactaa cttcagcctg 4381 ctgaagcagg ctggagacgt ggaggagaac cctggaccta gcgagctgat taaggagaac 4441 atgcacatga agctgtacat ggagggcacc gtggacaacc atcacttcaa gtgcacatcc 4501 gagggcgaag gcaagcccta cgagggcacc cagaccatga gaatcaaggt ggtcgagggc 4561 ggccctctcc ccttcgcctt cgacatcctg gctactagct tcctctacgg cagcaagacc 4621 ttcatcaacc acacccaggg catccccgac ttcttcaagc agtccttccc tgagggcttc 4681 acatgggaga gagtcaccac atacgaagac gggggcgtgc tgaccgctac ccaggacacc 4741 agcctccagg acggctgcct catctacaac gtcaagatca gaggggtgaa cttcacatcc 4801 aacggccctg tgatgcagaa gaaaacactc ggctgggagg ccttcaccga gacgctgtac 4861 cccgctgacg gcggcctgga aggcagaaac gacatggccc tgaagctcgt gggcgggagc 4921 catctgatcg caaacatcaa gaccacatat agatccaaga aacccgctaa gaacctcaag 4981 atgcctggcg tctactatgt ggactacaga ctggaaagaa tcaaggaggc caacaacgag 5041 acctacgtcg agcagcacga ggtggcagtg gccagatact gcgacctccc tagcaaactg 5101 gggcacaagc ttaattaaga attcgtcgag ggacctaata acttcgtata gcatacatta 5161 tacgaagtta tacatgttta agggttccgg ttccactagg tacaattcga tatcaagctt 5221 atcgataatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat 5281 gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct 5341 tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag 5401 gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc 5461 cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc 5521 ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct 5581 cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg 5641 ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg 5701 gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg 5761 cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcatcga 5821 taccgtcgac ctcgatcgag acctagaaaa acatggagca atcacaagta gcaatacagc 5881 agctaccaat gctgattgtg cctggctaga agcacaagag gaggaggagg tgggttttcc 5941 agtcacacct caggtacctt taagaccaat gacttacaag gcagctgtag atcttagcca 6001 ctttttaaaa gaaaaggggg gactggaagg gctaattcac tcccaacgaa gacaagatat 6061 ccttgatctg tggatctacc acacacaagg ctacttccct gattggcaga actacacacc 6121 agggccaggg atcagatatc cactgacctt tggatggtgc tacaagctag taccagttga 6181 gcaagagaag gtagaagaag ccaatgaagg agagaacacc cgcttgttac accctgtgag 6241 cctgcatggg atggatgacc cggagagaga agtattagag tggaggtttg acagccgcct 6301 agcatttcat cacatggccc gagagctgca tccggactgt actgggtctc tctggttaga 6361 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata 6421 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta 6481 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagcatgt gagcaaaagg 6541 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg 6601 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg 6661 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac 6721 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca 6781 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt 6841 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc 6901 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag 6961 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac 7021 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt 7081 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa 7141 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg 7201 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa 7261 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat 7321 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc 7381 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat 7441 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc 7501 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc 7561 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag 7621 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg 7681 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg 7741 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag 7801 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt 7861 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga 7921 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc 7981 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc 8041 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc 8101 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc 8161 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca 8221 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat 8281 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgac //