LOCUS Exported 8733 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION sgRNA (no RNA aptamer addition) with COM-KRAB effector for mammalian cells. ACCESSION . VERSION . KEYWORDS pJZC73 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8733) 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 8733) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8733 /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" enhancer 3175..3478 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 3479..3682 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" CDS 3719..3739 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 4004..4189 /codon_start=1 /product="Kruppel-associated box (KRAB) transcriptional repression domain from the human zinc finger protein ZNF10 (Margolin et al., 1994)" /label=KRAB /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY QLTKPDVILRLEKGEEP" misc_feature 4229..4802 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(4396..4413) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 4623..4642 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 4803..5513 /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA EGRHSTGGMDELYK" primer_bind complement(4948..4966) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 5216..5235 /label=mCherry-F /note="mCherry, forward primer" protein_bind 5533..5566 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 5622..6210 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(5675..5695) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(6093..6104) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(6213..6229) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6214..6230) /label=pBluescriptKS /note="For pBluescript vector" LTR 6739..6919 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" rep_origin complement(6981..7569) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(7061..7080) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(7740..8600) /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 8363..8382 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(8601..8705) /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 gttggaatag ctcagaggcc gagggtttaa gagctatgct ggaaacagca tagcaagttt 3001 aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc tttttttctc 3061 gagtactagg atccattagg cggccgcgtg gataaccgta ttaccgccat gcattagtta 3121 ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac 3181 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc 3241 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt 3301 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac 3361 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac 3421 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt 3481 gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc 3541 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt 3601 tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg 3661 ggaggtctat ataagcagag ctggtttagt gaaccgtcag atccgctagc ccaccatgcc 3721 caaaaagaaa agaaaagtgg gtagtatgaa atcaattcgc tgtaaaaact gcaacaaact 3781 gttatttaag gcggatagtt ttgatcacat tgaaatcagg tgtccgcgtt gcaaacgtca 3841 catcataatg ctgaatgcct gcgagcatcc cacggagaaa cattgtggga aaagagaaaa 3901 aatcacgcat tctgacgaaa ccgtgcgtta tggctccact agtgggcaca agcttaatgg 3961 cggtggcgga gggatggatg ctaagtcact aactgcctgg tcccggacac tggtgacctt 4021 caaggatgta tttgtggact tcaccaggga ggagtggaag ctgctggaca ctgctcagca 4081 gatcgtgtac agaaatgtga tgctggagaa ctataagaac ctggtttcct tgggttatca 4141 gcttactaag ccagatgtga tcctccggtt ggagaaggga gaagagccct agcccgggtc 4201 gagcatgcat ctagggcggc caattccgcc cctctccctc ccccccccct aacgttactg 4261 gccgaagccg cttggaataa ggccggtgtg cgtttgtcta tatgttattt tccaccatat 4321 tgccgtcttt tggcaatgtg agggcccgga aacctggccc tgtcttcttg acgagcattc 4381 ctaggggtct ttcccctctc gccaaaggaa tgcaaggtct gttgaatgtc gtgaaggaag 4441 cagttcctct ggaagcttct tgaagacaaa caacgtctgt agcgaccctt tgcaggcagc 4501 ggaacccccc acctggcgac aggtgcctct gcggccaaaa gccacgtgta taagatacac 4561 ctgcaaaggc ggcacaaccc cagtgccacg ttgtgagttg gatagttgtg gaaagagtca 4621 aatggctctc ctcaagcgta ttcaacaagg ggctgaagga tgcccagaag gtaccccatt 4681 gtatgggatc tgatctgggg cctcggtgca catgctttac atgtgtttag tcgaggttaa 4741 aaaaacgtct aggccccccg aaccacgggg acgtggtttt cctttgaaaa acacgatgat 4801 aaatggtgag caagggcgag gaggataaca tggccatcat caaggagttc atgcgcttca 4861 aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg 4921 gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc 4981 ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc 5041 accccgccga catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc 5101 gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg 5161 acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg 5221 taatgcagaa gaagaccatg ggctgggagg cctcctccga gcggatgtac cccgaggacg 5281 gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg 5341 ctgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg 5401 tcaacatcaa gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg 5461 aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtacaag taagaattcg 5521 tcgagggacc taataacttc gtatagcata cattatacga agttatacat gtttaagggt 5581 tccggttcca ctaggtacaa ttcgatatca agcttatcga taatcaacct ctggattaca 5641 aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat 5701 acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct 5761 ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac 5821 gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca 5881 cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca 5941 tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg 6001 tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga 6061 ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt 6121 cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga 6181 gtcggatctc cctttgggcc gcctccccgc atcgataccg tcgacctcga tcgagaccta 6241 gaaaaacatg gagcaatcac aagtagcaat acagcagcta ccaatgctga ttgtgcctgg 6301 ctagaagcac aagaggagga ggaggtgggt tttccagtca cacctcaggt acctttaaga 6361 ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg 6421 gaagggctaa ttcactccca acgaagacaa gatatccttg atctgtggat ctaccacaca 6481 caaggctact tccctgattg gcagaactac acaccagggc cagggatcag atatccactg 6541 acctttggat ggtgctacaa gctagtacca gttgagcaag agaaggtaga agaagccaat 6601 gaaggagaga acacccgctt gttacaccct gtgagcctgc atgggatgga tgacccggag 6661 agagaagtat tagagtggag gtttgacagc cgcctagcat ttcatcacat ggcccgagag 6721 ctgcatccgg actgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct 6781 ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta 6841 gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca 6901 gtgtggaaaa tctctagcag catgtgagca aaaggccagc aaaaggccag gaaccgtaaa 6961 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 7021 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 7081 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 7141 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt 7201 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 7261 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 7321 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 7381 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc 7441 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa 7501 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 7561 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac 7621 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta 7681 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt 7741 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata 7801 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc 7861 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac 7921 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag 7981 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac 8041 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc 8101 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg 8161 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc 8221 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct 8281 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc 8341 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc 8401 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc 8461 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc 8521 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca 8581 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt 8641 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt 8701 ccgcgcacat ttccccgaaa agtgccacct gac //