LOCUS Exported 7653 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION sgRNA for mammalian cells with mCherry marker. ACCESSION . VERSION . KEYWORDS pJZC35 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7653) 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 7653) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7653 /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 3176..3479 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 3480..3683 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" CDS 3726..4433 /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE GRHSTGGMDELYK" primer_bind complement(3868..3886) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 4136..4155 /label=mCherry-F /note="mCherry, forward primer" protein_bind 4453..4486 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 4542..5130 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(4595..4615) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(5013..5024) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(5133..5149) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5134..5150) /label=pBluescriptKS /note="For pBluescript vector" LTR 5659..5839 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" rep_origin complement(5901..6489) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5981..6000) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6660..7520) /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 7283..7302 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7521..7625) /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 gttggtacgt tctctatcac tgatagttta agagctatgc tggaaacagc atagcaagtt 3001 taaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttttttct 3061 cgagtactag gatccattag gcggccgcgt ggataaccgt attaccgcca tgcattagtt 3121 attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta 3181 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt 3241 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg 3301 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta 3361 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga 3421 ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg 3481 tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc 3541 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact 3601 ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt 3661 gggaggtcta tataagcaga gctggtttag tgaaccgtca gatccgctag cccaccatgc 3721 ccggggtgag caagggcgag gaggataaca tggccatcat caaggagttc atgcgcttca 3781 aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg 3841 gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc 3901 ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc 3961 accccgccga catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc 4021 gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg 4081 acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg 4141 taatgcagaa gaagaccatg ggctgggagg cctcctccga gcggatgtac cccgaggacg 4201 gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg 4261 ctgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg 4321 tcaacatcaa gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg 4381 aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtacaag taagaattcg 4441 tcgagggacc taataacttc gtatagcata cattatacga agttatacat gtttaagggt 4501 tccggttcca ctaggtacaa ttcgatatca agcttatcga taatcaacct ctggattaca 4561 aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat 4621 acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct 4681 ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac 4741 gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca 4801 cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca 4861 tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg 4921 tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga 4981 ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt 5041 cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga 5101 gtcggatctc cctttgggcc gcctccccgc atcgataccg tcgacctcga tcgagaccta 5161 gaaaaacatg gagcaatcac aagtagcaat acagcagcta ccaatgctga ttgtgcctgg 5221 ctagaagcac aagaggagga ggaggtgggt tttccagtca cacctcaggt acctttaaga 5281 ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg 5341 gaagggctaa ttcactccca acgaagacaa gatatccttg atctgtggat ctaccacaca 5401 caaggctact tccctgattg gcagaactac acaccagggc cagggatcag atatccactg 5461 acctttggat ggtgctacaa gctagtacca gttgagcaag agaaggtaga agaagccaat 5521 gaaggagaga acacccgctt gttacaccct gtgagcctgc atgggatgga tgacccggag 5581 agagaagtat tagagtggag gtttgacagc cgcctagcat ttcatcacat ggcccgagag 5641 ctgcatccgg actgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct 5701 ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta 5761 gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca 5821 gtgtggaaaa tctctagcag catgtgagca aaaggccagc aaaaggccag gaaccgtaaa 5881 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 5941 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 6001 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 6061 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt 6121 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 6181 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 6241 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 6301 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc 6361 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa 6421 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 6481 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac 6541 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta 6601 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt 6661 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata 6721 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc 6781 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac 6841 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag 6901 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac 6961 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc 7021 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg 7081 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc 7141 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct 7201 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc 7261 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc 7321 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc 7381 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc 7441 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca 7501 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt 7561 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt 7621 ccgcgcacat ttccccgaaa agtgccacct gac //