LOCUS Exported 8698 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Lentiviral vector for SAM-CRISPRa gRNA expression with puroBFP. ACCESSION . VERSION . KEYWORDS pKVL2-U6gRNA_SAM(BbsI)-PGKpuroBFP-W SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8698) AUTHORS Chong ZS, Ohnishi S, Yusa K, Wright GJ TITLE Pooled extracellular receptor-ligand interaction screening using CRISPR activation. JOURNAL Genome Biol. 2018 Nov 26;19(1):205. doi: 10.1186/s13059-018-1581-3. PUBMED 30477585 REFERENCE 2 (bases 1 to 8698) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8698 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin complement(3..458) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(140..161) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 352..371 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 585..607 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 599..616 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 600..616 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 626..645 /label=T7 /note="T7 promoter, forward primer" promoter 626..644 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" promoter 960..1158 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 1113..1133 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" LTR 1176..1356 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 1403..1528 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 2027..2260 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 2445..2489 /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 2787..2904 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 2979..3219 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 2979..2999 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 3150..3169 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 3263..3281 /label=MS2 stem loop /note="stem loop that binds the bacteriophage MS2 coat protein" misc_RNA 3333..3351 /label=MS2 stem loop /note="stem loop that binds the bacteriophage MS2 coat protein" promoter 3404..3903 /label=PGK promoter /note="mouse phosphoglycerate kinase 1 promoter" primer_bind complement(3439..3462) /label=MSCV-rev /note="Murine stem cell virus, reverse primer" primer_bind 3836..3855 /label=mPGK-F /note="Mouse PGK promoter, forward primer" CDS 3943..4539 /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(3943..3962) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 4439..4459 /label=Puro-F /note="Puromycin resistance gene, forward primer" CDS 4558..4611 /codon_start=1 /product="2A peptide from Thosea asigna virus capsid protein" /label=T2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="EGRGSLLTCGDVEENPGP" CDS 4612..5313 /codon_start=1 /product="monomeric blue fluorescent protein" /label=TagBFP /note="mammalian codon-optimized" /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC DLPSKLGHKLN" primer_bind complement(5373..5393) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(5791..5802) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(6254..6271) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 6260..6484 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(6510..6530) /label=T3 /note="T3 promoter, forward primer" promoter complement(6510..6528) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(6549..6565) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6549..6565) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(6562..6584) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 6573..6589 /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(6597..6627) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6642..6663 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(6780..6797) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6951..7539) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(7031..7050) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(7710..8570) /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 8333..8352 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(8571..8675) /gene="bla" /label=AmpR promoter ORIGIN 1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc 181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc 241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag 301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa 361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac 421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg 481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg 541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg 601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgactagtac 661 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc 721 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt 781 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac 841 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac 901 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt 961 gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc 1021 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt 1081 tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg 1141 ggaggtctat ataagcagcg cgttttgcct gtactgggtc tctctggtta gaccagatct 1201 gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc 1261 cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc 1321 tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa 1381 agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg aagcgcgcac 1441 ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta gcggaggcta 1501 gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta gatcgcgatg 1561 ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac atatagtatg 1621 ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg 1681 ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag aagaacttag 1741 atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag agataaaaga 1801 caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga ccaccgcaca 1861 gcaagcggcc ggccgcgctg atcttcagac ctggaggagg agatatgagg gacaattgga 1921 gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca 1981 aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga gctttgttcc 2041 ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac 2101 aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg agggctattg 2161 aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc caggcaagaa 2221 tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg ggttgctctg 2281 gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat aaatctctgg 2341 aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac aattacacaa 2401 gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat gaacaagaat 2461 tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca aattggctgt 2521 ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga atagtttttg 2581 ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg tttcagaccc 2641 acctcccaac cccgagggga cccgacaggc ccgaaggaat agaagaagaa ggtggagaga 2701 gagacagaga cagatccatt cgattagtga acggatcggc actgcgtgcg ccaattctgc 2761 agacaaatgg cagtattcat ccacaatttt aaaagaaaag gggggattgg ggggtacagt 2821 gcaggggaaa gaatagtaga cataatagca acagacatac aaactaaaga attacaaaaa 2881 caaattacaa aaattcaaaa ttttcgggtt tattacaggg acagcagaga tccagtttgg 2941 ttagtaccgg gccctacgcg tccaaggtcg ggcaggaaga gggcctattt cccatgattc 3001 cttcatattt gcatatacga tacaaggctg ttagagagat aattagaatt aatttgactg 3061 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt 3121 ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt 3181 atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccggg tcttcgagaa 3241 gacacgttta agagctaggc caacatgagg atcacccatg tctgcagggc ctagcaagtt 3301 taaataaggc tagtccgtta tcaacttggc caacatgagg atcacccatg tctgcagggc 3361 caagtggcac cgagtcggtg ctttttttgg atccaattct accgggtagg ggaggcgctt 3421 ttcccaaggc agtctggagc atgcgcttta gcagccccgc tgggcacttg gcgctacaca 3481 agtggcctct ggcctcgcac acattccaca tccaccggta ggcgccaacc ggctccgttc 3541 tttggtggcc ccttcgcgcc accttctact cctcccctag tcaggaagtt cccccccgcc 3601 ccgcagctcg cgtcgtgcag gacgtgacaa atggaagtag cacgtctcac tagtctcgtg 3661 cagatggaca gcaccgctga gcaatggaag cgggtaggcc tttggggcag cggccaatag 3721 cagctttgct ccttcgcttt ctgggctcag aggctgggaa ggggtgggtc cgggggcggg 3781 ctcaggggcg ggctcagggg cggggcgggc gcccgaaggt cctccggagg cccggcattc 3841 tgcacgcttc aaaagcgcac gtctgccgcg ctgttctcct cttcctcatc tccgggcctt 3901 tcgacctgca tccatctaga tctcgagcag ctgaagctta ccatgaccga gtacaagccc 3961 acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg 4021 ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc 4081 accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc 4141 gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg 4201 gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag 4261 caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc 4321 accgtcggcg tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc 4381 ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc 4441 aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa 4501 ggaccgcgca cctggtgcat gacccgcaag cccggtgccg gcggcgggtc cggaggagag 4561 ggcagaggaa gtctcctaac atgcggtgac gtggaggaga atcctggccc aatgagcgag 4621 ctgattaagg agaacatgca catgaagctg tacatggagg gcaccgtgga caaccatcac 4681 ttcaagtgca catccgaggg cgaaggcaag ccctacgagg gcacccagac catgagaatc 4741 aaggtggtcg agggcggccc tctccccttc gccttcgaca tcctggctac tagcttcctc 4801 tacggcagca agaccttcat caaccacacc cagggcatcc ccgacttctt caagcagtcc 4861 ttccctgagg gcttcacatg ggagagagtc accacatacg aggacggggg cgtgctgacc 4921 gctacccagg acaccagcct ccaggacggc tgcctcatct acaacgtcaa gatcagaggg 4981 gtgaacttca catccaacgg ccctgtgatg cagaagaaaa cactcggctg ggaggccttc 5041 accgagacgc tgtaccccgc tgacggcggc ctggaaggca gaaacgacat ggccctgaag 5101 ctcgtgggcg ggagccatct gatcgcaaac atcaagacca catatagatc caagaaaccc 5161 gctaagaacc tcaagatgcc tggcgtctac tatgtggact acagactgga aagaatcaag 5221 gaggccaaca acgagaccta cgtcgagcag cacgaggtgg cagtggccag atactgcgac 5281 ctccctagca aactggggca caagcttaat tgagcggccg ctcaacctct ggattacaaa 5341 atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac 5401 gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc 5461 ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt 5521 ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc 5581 tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc 5641 gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg 5701 gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt 5761 ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc 5821 cgcggcctgc tgccggctct gcggcctctt ccgcgtctac gccttcgccc tcagacgagt 5881 cggatctccc tttgggccgc ctccccgcag gtacctttaa gaccaatgac ttacaaggca 5941 gctgtagatc ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc 6001 caaagaagtc aagatctgct ttttgcctgt actgggtctc tctggttaga ccagagtctc 6061 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta 6121 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact 6181 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtttaa 6241 acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc 6301 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag 6361 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag 6421 gacagcaagg gggaggattg ggaagtcaat agcaggcatg ctggggatgc ggtgggctct 6481 atgggcggcc gttaattaag cttttgttcc ctttagtgag ggttaattgc gcgcttggcg 6541 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac 6601 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca 6661 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat 6721 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc 6781 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 6841 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 6901 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 6961 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 7021 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 7081 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 7141 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 7201 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 7261 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 7321 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 7381 tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 7441 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 7501 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 7561 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 7621 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 7681 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 7741 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact 7801 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc 7861 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt 7921 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta 7981 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg 8041 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt 8101 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc 8161 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt 8221 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc 8281 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 8341 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 8401 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 8461 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 8521 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 8581 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 8641 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccac //