LOCUS Exported 8714 bp ds-DNA circular SYN 14-DEC-2021 DEFINITION For cloning of guide RNAs libraries compatible with RfxCas13d. Contains a 5' modified direct repeat. Clone using BsmBI. F overhang aaac. R overhang aaaa. ACCESSION . VERSION . KEYWORDS pLentiRNAGuide_001 - hU6-RfxCas13d-DR1-BsmBI-EFS-Puro-WPRE SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8714) AUTHORS Wessels HH, Mendez-Mancilla A, Guo X, Legut M, Daniloski Z, Sanjana NE TITLE Massively parallel Cas13 screens reveal principles for guide RNA design. JOURNAL Nat Biotechnol. 2020 Jun;38(6):722-727. doi: 10.1038/s41587-020-0456-9. Epub 2020 Mar 16. PUBMED 32518401 REFERENCE 2 (bases 1 to 8714) AUTHORS . TITLE Direct Submission JOURNAL Exported Dec 14, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8714 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 1..380 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 382..580 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 535..555 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" LTR 598..778 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 825..950 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1443..1676 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 1861..1905 /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 2203..2320 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 2371..2611 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 2371..2391 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 2542..2561 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 2622..2650 /label=RfxCas13d DR30 /note="5' processed 30-base direct repeat for an RfxCas13d gRNA (Konermann et al., 2018)" promoter 2727..2938 /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" CDS 2954..3553 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind complement(2954..2973) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 3450..3470 /label=Puro-F /note="Puromycin resistance gene, forward primer" misc_feature 3569..4157 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(3622..3642) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(4040..4051) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 4229..4462 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" primer_bind complement(4488..4505) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 4494..4718 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 4764..5192 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4851..4870) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5061..5082 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(5201..5221) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 5206..5535 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 5386..5521 /label=SV40 ori /note="SV40 origin of replication" primer_bind 5448..5467 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 5583..5630 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 5649..6023 /codon_start=1 /gene="Sh ble from Streptoalloteichus hindustanus" /product="antibiotic-binding protein" /label=BleoR /note="confers resistance to bleomycin, phleomycin, and Zeocin(TM)" /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR EFALRDPAGNCVHFVAEEQD" polyA_signal 6153..6274 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(6190..6209) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 6244..6263 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(6323..6339) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6323..6339) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(6336..6358) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 6347..6363 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(6371..6401) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6416..6437 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(6554..6571) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6725..7313) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6805..6824) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(7484..8344) /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 8107..8126 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(8345..8449) /gene="bla" /label=AmpR promoter primer_bind complement(8524..8543) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc 421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa 541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag cgcgttttgc ctgtactggg 601 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg 661 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt 721 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt 781 ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga 841 ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa 901 aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc 961 gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat 1021 ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg 1081 gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc 1141 agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc 1201 atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa 1261 acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat 1321 atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta 1381 ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga 1441 ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca 1501 atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat 1561 ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag 1621 cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg 1681 atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg 1741 agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa 1801 attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa 1861 aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac 1921 ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt 1981 ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca 2041 ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa 2101 gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg atcggcactg 2161 cgtgcgccaa ttctgcagac aaatggcagt attcatccac aattttaaaa gaaaaggggg 2221 gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac 2281 taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag 2341 cagagatcca gtttggttaa ttaaggtacc gagggcctat ttcccatgat tccttcatat 2401 ttgcatatac gatacaaggc tgttagagag ataattggaa ttaatttgac tgtaaacaca 2461 aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt 2521 ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat 2581 ttcttggctt tatatatctt gtggaaagga cgaaacaccg tacccctacc aactggtcgg 2641 ggtttgaaac ggagacggac gtctcttttt tgaattcgct agctaggtct tgaaaggagt 2701 gggaattgac tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa 2761 gttgggggga ggggtcggca attgatccgg tgcctagaga aggtggcgcg gggtaaactg 2821 ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat 2881 aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacagga 2941 ccggttctag agcatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt 3001 ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac 3061 cgtcgatccg gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg 3121 cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg 3181 gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc 3241 cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca 3301 ccggcccaag gagcccgcgt ggttcctggc caccgtcgga gtctcgcccg accaccaggg 3361 caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt 3421 gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt 3481 caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa 3541 gcccggtgcc tgaacgcgtt aagtcgacaa tcaacctctg gattacaaaa tttgtgaaag 3601 attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat 3661 gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc 3721 ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg 3781 cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct 3841 ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct 3901 tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg 3961 gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac 4021 gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct 4081 gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct 4141 ttgggccgcc tccccgcgtc gactttaaga ccaatgactt acaaggcagc tgtagatctt 4201 agccactttt taaaagaaaa ggggggactg gaagggctaa ttcactccca acgaagacaa 4261 gatctgcttt ttgcttgtac tgggtctctc tggttagacc agatctgagc ctgggagctc 4321 tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa 4381 gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag 4441 tcagtgtgga aaatctctag cagggcccgt ttaaacccgc tgatcagcct cgactgtgcc 4501 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg 4561 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag 4621 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga 4681 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag 4741 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt 4801 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc 4861 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg 4921 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta 4981 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt 5041 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat 5101 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa 5161 tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg 5221 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag 5281 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg 5341 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact 5401 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag 5461 gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc 5521 ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcagcac 5581 gtgttgacaa ttaatcatcg gcatagtata tcggcatagt ataatacgac aaggtgagga 5641 actaaaccat ggccaagttg accagtgccg ttccggtgct caccgcgcgc gacgtcgccg 5701 gagcggtcga gttctggacc gaccggctcg ggttctcccg ggacttcgtg gaggacgact 5761 tcgccggtgt ggtccgggac gacgtgaccc tgttcatcag cgcggtccag gaccaggtgg 5821 tgccggacaa caccctggcc tgggtgtggg tgcgcggcct ggacgagctg tacgccgagt 5881 ggtcggaggt cgtgtccacg aacttccggg acgcctccgg gccggccatg accgagatcg 5941 gcgagcagcc gtgggggcgg gagttcgccc tgcgcgaccc ggccggcaac tgcgtgcact 6001 tcgtggccga ggagcaggac tgacacgtgc tacgagattt cgattccacc gccgccttct 6061 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg 6121 gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt 6181 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta 6241 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta 6301 gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca 6361 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag 6421 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt 6481 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc 6541 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg 6601 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa 6661 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 6721 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 6781 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 6841 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 6901 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc 6961 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 7021 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca 7081 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt 7141 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag 7201 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 7261 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc 7321 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt 7381 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt 7441 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca 7501 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg 7561 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac 7621 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg 7681 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc 7741 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta 7801 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac 7861 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc 7921 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac 7981 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact 8041 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa 8101 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt 8161 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca 8221 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa 8281 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac 8341 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg 8401 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc 8461 gaaaagtgcc acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct 8521 cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt 8581 ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga 8641 caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc 8701 cagatatacg cgtt //