LOCUS Exported 8323 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION 3rd generation lentiviral gRNA plasmid targeting human STK3. ACCESSION . VERSION . KEYWORDS STK3 gRNA (BRDN0001147084) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8323) AUTHORS Doench JG, Fusi N, Sullender M, Hegde M, Vaimberg EW, Donovan KF, Smith I, Tothova Z, Wilen C, Orchard R, Virgin HW, Listgarten J, Root DE TITLE Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. JOURNAL Nat Biotechnol. 2016 Jan 18. doi: 10.1038/nbt.3437. PUBMED 26780180 REFERENCE 2 (bases 1 to 8323) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8323 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 87..1265 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 318..1256 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 1213..1233 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" CDS 1278..1877 /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(1278..1297) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 1774..1794 /label=Puro-F /note="Puromycin resistance gene, forward primer" misc_feature 1893..2481 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(1946..1966) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(2364..2375) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 2553..2786 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 2864..2985 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2901..2920) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 2955..2974 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 3025..3160 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3087..3106 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(3180..3199) /label=T7 /note="T7 promoter, forward primer" promoter complement(3181..3199) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(3209..3226) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(3209..3225) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3218..3240) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 3367..3822 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3454..3473) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3664..3685 /label=F1ori-F /note="F1 origin, forward primer" promoter 3848..3952 /gene="bla" /label=AmpR promoter CDS 3953..4813 /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(4171..4190) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 4984..5572 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5473..5492 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 5726..5743 /label=L4440 /note="L4440 vector, forward primer" protein_bind 5860..5881 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 5896..5926 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5934..5950 /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 5939..5961 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 5958..5974 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 5958..5974 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 5993..6013 /label=T3 /note="T3 promoter, forward primer" promoter 5995..6013 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 6041..6267 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 6268..6448 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 6495..6620 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 7113..7346 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 7531..7575 /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 7753..7993 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 7753..7773 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 7924..7943 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 8023..8098 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" misc_feature 8154..8271 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" ORIGIN 1 cccgggtgca aagatggata aagttttaaa cagagaggaa tctttgcagc taatggacct 61 tctaggtctt gaaaggagtg ggaattggct ccggtgcccg tcagtgggca gagcgcacat 121 cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgatccggt gcctagagaa 181 ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg 241 gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt 301 ttgccgccag aacacaggta agtgccgtgt gtggttcccg cgggcctggc ctctttacgg 361 gttatggccc ttgcgtgcct tgaattactt ccacctggct gcagtacgtg attcttgatc 421 ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc ttgcgcttaa ggagcccctt 481 cgcctcgtgc ttgagttgag gcctggcctg ggcgctgggg ccgccgcgtg cgaatctggt 541 ggcaccttcg cgcctgtctc gctgctttcg ataagtctct agccatttaa aatttttgat 601 gacctgctgc gacgcttttt ttctggcaag atagtcttgt aaatgcgggc caagatctgc 661 acactggtat ttcggttttt ggggccgcgg gcggcgacgg ggcccgtgcg tcccagcgca 721 catgttcggc gaggcggggc ctgcgagcgc ggccaccgag aatcggacgg gggtagtctc 781 aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc gtgtatcgcc ccgccctggg 841 cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga aagatggccg cttcccggcc 901 ctgctgcagg gagctcaaaa tggaggacgc ggcgctcggg agagcgggcg ggtgagtcac 961 ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc ttcatgtgac tccacggagt 1021 accgggcgcc gtccaggcac ctcgattagt tctcgagctt ttggagtacg tcgtctttag 1081 gttgggggga ggggttttat gcgatggagt ttccccacac tgagtgggtg gagactgaag 1141 ttaggccagc ttggcacttg atgtaattct ccttggaatt tgcccttttt gagtttggat 1201 cttggttcat tctcaagcct cagacagtgg ttcaaagttt ttttcttcca tttcaggtgt 1261 cgtgacgtac ggccaccatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg 1321 acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc 1381 acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca 1441 cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gccgtggcgg 1501 tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca 1561 tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc 1621 cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggagtctcg cccgaccacc 1681 agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg 1741 gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg 1801 gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc 1861 gcaagcccgg tgcctgaacg cgttaagtcg acaatcaacc tctggattac aaaatttgtg 1921 aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt 1981 taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata 2041 aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg 2101 tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc 2161 tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct 2221 gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt 2281 cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg 2341 ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc 2401 tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct 2461 ccctttgggc cgcctccccg cgtcgacttt aagaccaatg acttacaagg cagctgtaga 2521 tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact cccaacgaag 2581 acaagatctg ctttttgctt gtactgggtc tctctggtta gaccagatct gagcctggga 2641 gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct 2701 tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt 2761 ttagtcagtg tggaaaatct ctagcagtac gtatagtagt tcatgtcatc ttattattca 2821 gtatttataa cttgcaaaga aatgaatatc agagagtgag aggaacttgt ttattgcagc 2881 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc 2941 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggctcta 3001 gctatcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct 3061 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct 3121 gagctattcc agaagtagtg aggaggcttt tttggaggcc tagggacgta cccaattcgc 3181 cctatagtga gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg 3241 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc 3301 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg 3361 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg 3421 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc 3481 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc 3541 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta 3601 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta 3661 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg 3721 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa 3781 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg 3841 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct 3901 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat 3961 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc 4021 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg 4081 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg 4141 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga 4201 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta 4261 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc 4321 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc 4381 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg 4441 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc 4501 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca 4561 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct 4621 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat 4681 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg 4741 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat 4801 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact 4861 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat 4921 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc 4981 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 5041 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg 5101 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca 5161 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc 5221 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 5281 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 5341 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga 5401 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 5461 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 5521 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 5581 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc 5641 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc 5701 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc 5761 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag 5821 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca 5881 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag 5941 cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa 6001 ccctcactaa agggaacaaa agctggagct gcaagcttaa tgtagtctta tgcaatactc 6061 ttgtagtctt gcaacatggt aacgatgagt tagcaacatg ccttacaagg agagaaaaag 6121 caccgtgcat gccgattggt ggaagtaagg tggtacgatc gtgccttatt aggaaggcaa 6181 cagacgggtc tgacatggat tggacgaacc actgaattgc cgcattgcag agatattgta 6241 tttaagtgcc tagctcgata cataaacggg tctctctggt tagaccagat ctgagcctgg 6301 gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg 6361 cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc 6421 ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa cagggacttg aaagcgaaag 6481 ggaaaccaga ggagctctct cgacgcagga ctcggcttgc tgaagcgcgc acggcaagag 6541 gcgaggggcg gcgactggtg agtacgccaa aaattttgac tagcggaggc tagaaggaga 6601 gagatgggtg cgagagcgtc agtattaagc gggggagaat tagatcgcga tgggaaaaaa 6661 ttcggttaag gccaggggga aagaaaaaat ataaattaaa acatatagta tgggcaagca 6721 gggagctaga acgattcgca gttaatcctg gcctgttaga aacatcagaa ggctgtagac 6781 aaatactggg acagctacaa ccatcccttc agacaggatc agaagaactt agatcattat 6841 ataatacagt agcaaccctc tattgtgtgc atcaaaggat agagataaaa gacaccaagg 6901 aagctttaga caagatagag gaagagcaaa acaaaagtaa gaccaccgca cagcaagcgg 6961 ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag tgaattatat 7021 aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc aaagagaaga 7081 gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg gttcttggga 7141 gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc cagacaatta 7201 ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc gcaacagcat 7261 ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct ggctgtggaa 7321 agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa actcatttgc 7381 accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca gatttggaat 7441 cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt aatacactcc 7501 ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt ggaattagat 7561 aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta tataaaatta 7621 ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt actttctata 7681 gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct cccaaccccg 7741 aggggaccca gagagggcct atttcccatg attccttcat atttgcatat acgatacaag 7801 gctgttagag agataattag aattaatttg actgtaaaca caaagatatt agtacaaaat 7861 acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa 7921 atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc 7981 ttgtggaaag gacgaaacac cgtggattgt tatggagtac tggttttaga gctagaaata 8041 gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt 8101 ttttaagctt ggcgtaacta gatcttgaga caaatggcag tattcatcca caattttaaa 8161 agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca 8221 gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat 8281 tacagggaca gcagagatcc actttggcgc cggctcgagg ggg //