LOCUS Exported 8332 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION U6 promoter expresses customizable Spyo-guide; EF1a promoter provides puromycin resistance. This vector is a derivative of the lentiGuide vector, with minor modifications to the tracrR. ACCESSION . VERSION . KEYWORDS pRDA_118 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8332) AUTHORS DeWeirdt PC, Sangree AK, Hanna RE, Sanson KR, Hegde M, Strand C, Persky NS, Doench JG TITLE Genetic screens in isogenic mammalian cell lines without single cell cloning. JOURNAL Nat Commun. 2020 Feb 6;11(1):752. doi: 10.1038/s41467-020-14620-6. PUBMED 32029722 REFERENCE 2 (bases 1 to 8332) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8332 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 1..227 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 228..408 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 455..580 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1073..1306 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 1491..1535 /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" promoter 1716..1956 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 1716..1736 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 1887..1906 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 1986..2061 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" misc_feature 2123..2240 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 2379..3557 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 2610..3548 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 3505..3525 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" CDS 3570..4169 /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(3570..3589) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 4066..4086 /label=Puro-F /note="Puromycin resistance gene, forward primer" misc_feature 4185..4773 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(4238..4258) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(4656..4667) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 4845..5078 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 5156..5277 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5193..5212) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 5247..5266 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 5317..5452 /label=SV40 ori /note="SV40 origin of replication" primer_bind 5379..5398 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(5472..5491) /label=T7 /note="T7 promoter, forward primer" promoter complement(5473..5491) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(5501..5518) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(5501..5517) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5510..5532) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 5659..6114 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5746..5765) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5956..5977 /label=F1ori-F /note="F1 origin, forward primer" promoter 6140..6244 /gene="bla" /label=AmpR promoter CDS 6245..7105 /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 complement(6463..6482) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7276..7864 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7765..7784 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 8018..8035 /label=L4440 /note="L4440 vector, forward primer" protein_bind 8152..8173 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 8188..8218 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 8226..8242 /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)." primer_bind 8231..8253 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 8250..8266 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 8250..8266 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 8285..8305 /label=T3 /note="T3 promoter, forward primer" promoter 8287..8305 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" ORIGIN 1 tgtagtctta tgcaatactc ttgtagtctt gcaacatggt aacgatgagt tagcaacatg 61 ccttacaagg agagaaaaag caccgtgcat gccgattggt ggaagtaagg tggtacgatc 121 gtgccttatt aggaaggcaa cagacgggtc tgacatggat tggacgaacc actgaattgc 181 cgcattgcag agatattgta tttaagtgcc tagctcgata cataaacggg tctctctggt 241 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc 301 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta 361 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa 421 cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga ctcggcttgc 481 tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa aaattttgac 541 tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc gggggagaat 601 tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa 661 acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga 721 aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc 781 agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat 841 agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa 901 gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat atgagggaca 961 attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac 1021 ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt 1081 tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga 1141 cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg 1201 ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg 1261 caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt 1321 gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat 1381 ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt 1441 acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac 1501 aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt 1561 ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag 1621 tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc 1681 agacccacct cccaaccccg aggggacccg gtaccgaggg cctatttccc atgattcctt 1741 catatttgca tatacgatac aaggctgtta gagagataat tagaattaat ttgactgtaa 1801 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg 1861 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt 1921 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccggagac gcttttttcg 1981 tctcagtttg agagctagaa atagcaagtt caaataaggc tagtccgtta tcaacttgaa 2041 aaagtggcac cgagtcggtg cttttttgaa ttcaagcttg gcgtaactag atcttgagac 2101 aaatggcagt attcatccac aattttaaaa gaaaaggggg gattgggggg tacagtgcag 2161 gggaaagaat agtagacata atagcaacag acatacaaac taaagaatta caaaaacaaa 2221 ttacaaaaat tcaaaatttt cgggtttatt acagggacag cagagatcca ctttggcgcc 2281 ggctcgaggg ggcccgggtg caaagatgga taaagtttta aacagagagg aatctttgca 2341 gctaatggac cttctaggtc ttgaaaggag tgggaattgg ctccggtgcc cgtcagtggg 2401 cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc aattgatccg 2461 gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc 2521 tttttcccga gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt 2581 ttcgcaacgg gtttgccgcc agaacacagg taagtgccgt gtgtggttcc cgcgggcctg 2641 gcctctttac gggttatggc ccttgcgtgc cttgaattac ttccacctgg ctgcagtacg 2701 tgattcttga tcccgagctt cgggttggaa gtgggtggga gagttcgagg ccttgcgctt 2761 aaggagcccc ttcgcctcgt gcttgagttg aggcctggcc tgggcgctgg ggccgccgcg 2821 tgcgaatctg gtggcacctt cgcgcctgtc tcgctgcttt cgataagtct ctagccattt 2881 aaaatttttg atgacctgct gcgacgcttt ttttctggca agatagtctt gtaaatgcgg 2941 gccaagatct gcacactggt atttcggttt ttggggccgc gggcggcgac ggggcccgtg 3001 cgtcccagcg cacatgttcg gcgaggcggg gcctgcgagc gcggccaccg agaatcggac 3061 gggggtagtc tcaagctggc cggcctgctc tggtgcctgg cctcgcgccg ccgtgtatcg 3121 ccccgccctg ggcggcaagg ctggcccggt cggcaccagt tgcgtgagcg gaaagatggc 3181 cgcttcccgg ccctgctgca gggagctcaa aatggaggac gcggcgctcg ggagagcggg 3241 cgggtgagtc acccacacaa aggaaaaggg cctttccgtc ctcagccgtc gcttcatgtg 3301 actccacgga gtaccgggcg ccgtccaggc acctcgatta gttctcgagc ttttggagta 3361 cgtcgtcttt aggttggggg gaggggtttt atgcgatgga gtttccccac actgagtggg 3421 tggagactga agttaggcca gcttggcact tgatgtaatt ctccttggaa tttgcccttt 3481 ttgagtttgg atcttggttc attctcaagc ctcagacagt ggttcaaagt ttttttcttc 3541 catttcaggt gtcgtgacgt acggccacca tgaccgagta caagcccacg gtgcgcctcg 3601 ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc gccgactacc 3661 ccgccacgcg ccacaccgtc gatccggacc gccacatcga gcgggtcacc gagctgcaag 3721 aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg 3781 ccgccgtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga 3841 tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa cagatggaag 3901 gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc gtcggagtct 3961 cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg 4021 ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac ctccccttct 4081 acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct 4141 ggtgcatgac ccgcaagccc ggtgcctgaa cgcgttaagt cgacaatcaa cctctggatt 4201 acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg 4261 gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct 4321 cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc 4381 aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca 4441 ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac 4501 tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt 4561 ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct 4621 ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc 4681 cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga 4741 cgagtcggat ctccctttgg gccgcctccc cgcgtcgact ttaagaccaa tgacttacaa 4801 ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca 4861 ctcccaacga agacaagatc tgctttttgc ttgtactggg tctctctggt tagaccagat 4921 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt 4981 gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc 5041 cctcagaccc ttttagtcag tgtggaaaat ctctagcagt acgtatagta gttcatgtca 5101 tcttattatt cagtatttat aacttgcaaa gaaatgaata tcagagagtg agaggaactt 5161 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa 5221 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca 5281 tgtctggctc tagctatccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt 5341 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc 5401 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctagggacg 5461 tacccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac 5521 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt 5581 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca 5641 gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg 5701 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct 5761 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc 5821 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg 5881 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt 5941 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg 6001 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc 6061 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg 6121 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa 6181 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa 6241 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct 6301 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg 6361 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg 6421 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt 6481 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga 6541 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga 6601 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac 6661 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg 6721 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac 6781 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct 6841 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct 6901 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg 6961 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat 7021 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg 7081 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat 7141 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct 7201 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa 7261 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa 7321 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc 7381 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta 7441 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct 7501 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg 7561 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag 7621 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc 7681 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg 7741 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt 7801 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg 7861 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca 7921 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg 7981 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc 8041 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag 8101 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag 8161 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg 8221 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa 8281 gcgcgcaatt aaccctcact aaagggaaca aaagctggag ctgcaagctt aa //