LOCUS Exported 7477 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Lentiviral expression of AAVS1-targeting sgRNA; insert can be replaced with custom sgRNA (see protocol). ACCESSION . VERSION . KEYWORDS pLX-sgRNA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7477) AUTHORS Wang T, Wei JJ, Sabatini DM, Lander ES TITLE Genetic screens in human cells using the CRISPR-Cas9 system. JOURNAL Science. 2014 Jan 3;343(6166):80-4. doi: 10.1126/science.1246981. Epub 2013 Dec 12. PUBMED 24336569 REFERENCE 2 (bases 1 to 7477) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7477 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 5..231 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 232..412 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 459..584 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1077..1310 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 1495..1539 /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 1714..2214 /label=hPGK promoter /note="human phosphoglycerate kinase 1 promoter" primer_bind 2133..2151 /label=hPGK-F /note="Human PGK promoter, forward primer" CDS 2226..2621 /codon_start=1 /gene="Aspergillus terreus BSD" /product="blasticidin S deaminase" /label=BSD /note="confers resistance to blasticidin" /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI KAIVKDSDGQPTAVGIRELLPSGYVWEG" misc_feature 2681..2798 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 2916..3156 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 2916..2936 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 3087..3106 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 3185..3260 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" protein_bind complement(3272..3296) /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" misc_feature 3334..3922 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(3387..3407) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3805..3816) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 3994..4227 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 4305..4426 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4342..4361) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4396..4415 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 4466..4601 /label=SV40 ori /note="SV40 origin of replication" primer_bind 4528..4547 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(4621..4640) /label=T7 /note="T7 promoter, forward primer" promoter complement(4622..4640) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(4650..4667) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(4650..4666) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4659..4681) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 4808..5263 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4895..4914) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5105..5126 /label=F1ori-F /note="F1 origin, forward primer" promoter 5289..5393 /gene="bla" /label=AmpR promoter CDS 5394..6254 /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(5612..5631) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6425..7013 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6914..6933 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 7167..7184 /label=L4440 /note="L4440 vector, forward primer" protein_bind 7301..7322 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 7337..7367 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 7375..7391 /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 7380..7402 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 7399..7415 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 7399..7415 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 7434..7454 /label=T3 /note="T3 promoter, forward primer" promoter 7436..7454 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" ORIGIN 1 ttaatgtagt cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa 61 catgccttac aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac 121 gatcgtgcct tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa 181 ttgccgcatt gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc 241 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag 301 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct 361 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc 421 cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc 481 ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt 541 tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga 601 gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat 661 taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt 721 tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag 781 gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa 841 ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa 901 gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg 961 gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta 1021 gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga 1081 gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg 1141 ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg 1201 agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc 1261 caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg 1321 ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat 1381 aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac 1441 aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat 1501 gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca 1561 aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga 1621 atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg 1681 tttcagaccc acctcccaac cccgagggga cccttgcgcc ttttccaagg cagccctggg 1741 tttgcgcagg gacgcggctg ctctgggcgt ggttccggga aacgcagcgg cgccgaccct 1801 gggtctcgca cattcttcac gtccgttcgc agcgtcaccc ggatcttcgc cgctaccctt 1861 gtgggccccc cggcgacgct tcctgctccg cccctaagtc gggaaggttc cttgcggttc 1921 gcggcgtgcc ggacgtgaca aacggaagcc gcacgtctca ctagtaccct cgcagacgga 1981 cagcgccagg gagcaatggc agcgcgccga ccgcgatggg ctgtggccaa tagcggctgc 2041 tcagcagggc gcgccgagag cagcggccgg gaaggggcgg tgcgggaggc ggggtgtggg 2101 gcggtagtgt gggccctgtt cctgcccgcg cggtgttccg cattctgcaa gcctccggag 2161 cgcacgtcgg cagtcggctc cctcgttgac cgaatcaccg acctctctcc ccagggggta 2221 ccaccatggc caagcctttg tctcaagaag aatccaccct cattgaaaga gcaacggcta 2281 caatcaacag catccccatc tctgaagact acagcgtcgc cagcgcagct ctctctagcg 2341 acggccgcat cttcactggt gtcaatgtat atcattttac tgggggacct tgtgcagaac 2401 tcgtggtgct gggcactgct gctgctgcgg cagctggcaa cctgacttgt atcgtcgcga 2461 tcggaaatga gaacaggggc atcttgagcc cctgcggacg gtgccgacag gtgcttctcg 2521 atctgcatcc tgggatcaaa gccatagtga aggacagtga tggacagccg acggcagttg 2581 ggattcgtga attgctgccc tctggttatg tgtgggaggg cctgcagctg cagtagtaag 2641 aattctagat cttgagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga 2701 ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta 2761 aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca 2821 gagatccact ttggcgccgg ctcgagtgta caaaaaagca ggctttaaag gaaccaattc 2881 agtcgactgg atccggtacc aaggtcgggc aggaagaggg cctatttccc atgattcctt 2941 catatttgca tatacgatac aaggctgtta gagagataat tagaattaat ttgactgtaa 3001 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg 3061 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt 3121 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccggggcc actagggaca 3181 ggatgtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa 3241 aagtggcacc gagtcggtgc tttttttcta gacccagctt tcttgtacaa agttggcatt 3301 agctagcgct aaccggtggc gcgttaagtc gacaatcaac ctctggatta caaaatttgt 3361 gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct 3421 ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat 3481 aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg 3541 gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag 3601 ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc 3661 tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg 3721 tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc 3781 gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc 3841 ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc 3901 tccctttggg ccgcctcccc gcgtcgactt taagaccaat gacttacaag gcagctgtag 3961 atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctaattcac tcccaacgaa 4021 gacaagatct gctttttgct tgtactgggt ctctctggtt agaccagatc tgagcctggg 4081 agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc 4141 ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct 4201 tttagtcagt gtggaaaatc tctagcagta cgtatagtag ttcatgtcat cttattattc 4261 agtatttata acttgcaaag aaatgaatat cagagagtga gaggaacttg tttattgcag 4321 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt 4381 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggctct 4441 agctatcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc 4501 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc 4561 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctagggacgt acccaattcg 4621 ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg 4681 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg 4741 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc 4801 gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc 4861 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt 4921 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc 4981 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt 5041 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt 5101 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt 5161 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa 5221 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg 5281 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc 5341 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta 5401 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg 5461 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg 5521 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac 5581 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg 5641 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt 5701 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg 5761 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac 5821 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt 5881 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag 5941 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc 6001 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc 6061 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta 6121 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg 6181 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga 6241 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac 6301 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa 6361 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat 6421 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc 6481 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg 6541 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc 6601 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg 6661 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg 6721 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa 6781 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg 6841 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga 6901 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct 6961 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca 7021 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc 7081 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg 7141 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc 7201 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca 7261 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc 7321 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga 7381 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag cgcgcaatta 7441 accctcacta aagggaacaa aagctggagc tgcaagc //