LOCUS Exported 8786 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION sgRNA + 1x COM with COM-KRAB effector for mammalian cells. ACCESSION . VERSION . KEYWORDS pJZC78 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8786) 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 8786) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8786 /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" terminator 3077..3096 /label=SUP4 terminator /note="transcription terminator for the S. cerevisiae SUP4 tRNA gene" enhancer 3228..3531 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 3532..3735 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" CDS 3772..3792 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 4057..4242 /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 4282..4855 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(4449..4466) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 4676..4695 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 4856..5566 /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(5001..5019) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 5269..5288 /label=mCherry-F /note="mCherry, forward primer" protein_bind 5586..5619 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 5675..6263 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(5728..5748) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(6146..6157) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(6266..6282) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6267..6283) /label=pBluescriptKS /note="For pBluescript vector" LTR 6792..6972 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" rep_origin complement(7034..7622) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(7114..7133) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(7793..8653) /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 8416..8435 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(8654..8758) /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 gttggcatac ttctgcctgc tggggagcct ggtttaagag ctatgctgga aacagcatag 3001 caagtttaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcctg 3061 aatgcctgcg agcatctttt tttgtttttt atgtctctgc agagttcggt ctcgagtact 3121 aggatccatt aggcggccgc gtggataacc gtattaccgc catgcattag ttattaatag 3181 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt 3241 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg 3301 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat 3361 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct 3421 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg 3481 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg 3541 ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc 3601 caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa 3661 tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc 3721 tatataagca gagctggttt agtgaaccgt cagatccgct agcccaccat gcccaaaaag 3781 aaaagaaaag tgggtagtat gaaatcaatt cgctgtaaaa actgcaacaa actgttattt 3841 aaggcggata gttttgatca cattgaaatc aggtgtccgc gttgcaaacg tcacatcata 3901 atgctgaatg cctgcgagca tcccacggag aaacattgtg ggaaaagaga aaaaatcacg 3961 cattctgacg aaaccgtgcg ttatggctcc actagtgggc acaagcttaa tggcggtggc 4021 ggagggatgg atgctaagtc actaactgcc tggtcccgga cactggtgac cttcaaggat 4081 gtatttgtgg acttcaccag ggaggagtgg aagctgctgg acactgctca gcagatcgtg 4141 tacagaaatg tgatgctgga gaactataag aacctggttt ccttgggtta tcagcttact 4201 aagccagatg tgatcctccg gttggagaag ggagaagagc cctagcccgg gtcgagcatg 4261 catctagggc ggccaattcc gcccctctcc ctcccccccc cctaacgtta ctggccgaag 4321 ccgcttggaa taaggccggt gtgcgtttgt ctatatgtta ttttccacca tattgccgtc 4381 ttttggcaat gtgagggccc ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg 4441 tctttcccct ctcgccaaag gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc 4501 tctggaagct tcttgaagac aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc 4561 cccacctggc gacaggtgcc tctgcggcca aaagccacgt gtataagata cacctgcaaa 4621 ggcggcacaa ccccagtgcc acgttgtgag ttggatagtt gtggaaagag tcaaatggct 4681 ctcctcaagc gtattcaaca aggggctgaa ggatgcccag aaggtacccc attgtatggg 4741 atctgatctg gggcctcggt gcacatgctt tacatgtgtt tagtcgaggt taaaaaaacg 4801 tctaggcccc ccgaaccacg gggacgtggt tttcctttga aaaacacgat gataaatggt 4861 gagcaagggc gaggaggata acatggccat catcaaggag ttcatgcgct tcaaggtgca 4921 catggagggc tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc 4981 ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc 5041 ctgggacatc ctgtcccctc agttcatgta cggctccaag gcctacgtga agcaccccgc 5101 cgacatcccc gactacttga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat 5161 gaacttcgag gacggcggcg tggtgaccgt gacccaggac tcctccctgc aggacggcga 5221 gttcatctac aaggtgaagc tgcgcggcac caacttcccc tccgacggcc ccgtaatgca 5281 gaagaagacc atgggctggg aggcctcctc cgagcggatg taccccgagg acggcgccct 5341 gaagggcgag atcaagcaga ggctgaagct gaaggacggc ggccactacg acgctgaggt 5401 caagaccacc tacaaggcca agaagcccgt gcagctgccc ggcgcctaca acgtcaacat 5461 caagttggac atcacctccc acaacgagga ctacaccatc gtggaacagt acgaacgcgc 5521 cgagggccgc cactccaccg gcggcatgga cgagctgtac aagtaagaat tcgtcgaggg 5581 acctaataac ttcgtatagc atacattata cgaagttata catgtttaag ggttccggtt 5641 ccactaggta caattcgata tcaagcttat cgataatcaa cctctggatt acaaaatttg 5701 tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc 5761 tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta 5821 taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt 5881 ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca 5941 gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc 6001 ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt 6061 gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct ggattctgcg 6121 cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg 6181 cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat 6241 ctccctttgg gccgcctccc cgcatcgata ccgtcgacct cgatcgagac ctagaaaaac 6301 atggagcaat cacaagtagc aatacagcag ctaccaatgc tgattgtgcc tggctagaag 6361 cacaagagga ggaggaggtg ggttttccag tcacacctca ggtaccttta agaccaatga 6421 cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc 6481 taattcactc ccaacgaaga caagatatcc ttgatctgtg gatctaccac acacaaggct 6541 acttccctga ttggcagaac tacacaccag ggccagggat cagatatcca ctgacctttg 6601 gatggtgcta caagctagta ccagttgagc aagagaaggt agaagaagcc aatgaaggag 6661 agaacacccg cttgttacac cctgtgagcc tgcatgggat ggatgacccg gagagagaag 6721 tattagagtg gaggtttgac agccgcctag catttcatca catggcccga gagctgcatc 6781 cggactgtac tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac 6841 tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg 6901 cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga 6961 aaatctctag cagcatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg 7021 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct 7081 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa 7141 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc 7201 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt 7261 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg 7321 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg 7381 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct 7441 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc 7501 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg 7561 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc 7621 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt 7681 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa 7741 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat 7801 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct 7861 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg 7921 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag 7981 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta 8041 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg 8101 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg 8161 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct 8221 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta 8281 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg 8341 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc 8401 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg 8461 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga 8521 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg 8581 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat 8641 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc 8701 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca 8761 catttccccg aaaagtgcca cctgac //