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" promoter 1..241 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 1..21 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 172..191 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 285..303 /label=MS2 stem loop /note="stem loop that binds the bacteriophage MS2 coat protein" misc_RNA 355..373 /label=MS2 stem loop /note="stem loop that binds the bacteriophage MS2 coat protein" promoter 426..925 /label=PGK promoter /note="mouse phosphoglycerate kinase 1 promoter" primer_bind complement(461..484) /label=MSCV-rev /note="Murine stem cell virus, reverse primer" primer_bind 858..877 /label=mPGK-F /note="Mouse PGK promoter, forward primer" CDS 965..1561 /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(965..984) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 1461..1481 /label=Puro-F /note="Puromycin resistance gene, forward primer" CDS 1580..1633 /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 1634..2335 /codon_start=1 /product="monomeric blue fluorescent protein" /label=TagBFP /note="mammalian codon-optimized" /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC DLPSKLGHKLN" primer_bind complement(2395..2415) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(2813..2824) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(3276..3293) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 3282..3506 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(3532..3552) /label=T3 /note="T3 promoter, forward primer" promoter complement(3532..3550) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(3571..3587) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3571..3587) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(3584..3606) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 3595..3611 /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(3619..3649) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3664..3685 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(3802..3819) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(3973..4561) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4053..4072) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4732..5592) /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 5355..5374 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5593..5697) /gene="bla" /label=AmpR promoter rep_origin complement(5723..6178) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5860..5881) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 6072..6091 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 6305..6327 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 6319..6336 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 6320..6336 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 6346..6365 /label=T7 /note="T7 promoter, forward primer" promoter 6346..6364 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" promoter 6680..6878 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 6833..6853 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" LTR 6896..7076 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 7123..7248 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 7747..7980 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 8165..8209 /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 8507..8624 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" ORIGIN 1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 61 ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 241 cgaaacaccg ggtcttcgag aagacacgtt taagagctag gccaacatga ggatcaccca 301 tgtctgcagg gcctagcaag tttaaataag gctagtccgt tatcaacttg gccaacatga 361 ggatcaccca tgtctgcagg gccaagtggc accgagtcgg tgcttttttt ggatccaatt 421 ctaccgggta ggggaggcgc ttttcccaag gcagtctgga gcatgcgctt tagcagcccc 481 gctgggcact tggcgctaca caagtggcct ctggcctcgc acacattcca catccaccgg 541 taggcgccaa ccggctccgt tctttggtgg ccccttcgcg ccaccttcta ctcctcccct 601 agtcaggaag ttcccccccg ccccgcagct cgcgtcgtgc aggacgtgac aaatggaagt 661 agcacgtctc actagtctcg tgcagatgga cagcaccgct gagcaatgga agcgggtagg 721 cctttggggc agcggccaat agcagctttg ctccttcgct ttctgggctc agaggctggg 781 aaggggtggg tccgggggcg ggctcagggg cgggctcagg ggcggggcgg gcgcccgaag 841 gtcctccgga ggcccggcat tctgcacgct tcaaaagcgc acgtctgccg cgctgttctc 901 ctcttcctca tctccgggcc tttcgacctg catccatcta gatctcgagc agctgaagct 961 taccatgacc gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc 1021 cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc 1081 ggaccgccac atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct 1141 cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc 1201 ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag 1261 cggttcccgg ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa 1321 ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct 1381 gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt 1441 cctggagacc tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac 1501 cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc 1561 cggcggcggg tccggaggag agggcagagg aagtctccta acatgcggtg acgtggagga 1621 gaatcctggc ccaatgagcg agctgattaa ggagaacatg cacatgaagc tgtacatgga 1681 gggcaccgtg gacaaccatc acttcaagtg cacatccgag ggcgaaggca agccctacga 1741 gggcacccag accatgagaa tcaaggtggt cgagggcggc cctctcccct tcgccttcga 1801 catcctggct actagcttcc tctacggcag caagaccttc atcaaccaca cccagggcat 1861 ccccgacttc ttcaagcagt ccttccctga gggcttcaca tgggagagag tcaccacata 1921 cgaggacggg ggcgtgctga ccgctaccca ggacaccagc ctccaggacg gctgcctcat 1981 ctacaacgtc aagatcagag gggtgaactt cacatccaac ggccctgtga tgcagaagaa 2041 aacactcggc tgggaggcct tcaccgagac gctgtacccc gctgacggcg gcctggaagg 2101 cagaaacgac atggccctga agctcgtggg cgggagccat ctgatcgcaa acatcaagac 2161 cacatataga tccaagaaac ccgctaagaa cctcaagatg cctggcgtct actatgtgga 2221 ctacagactg gaaagaatca aggaggccaa caacgagacc tacgtcgagc agcacgaggt 2281 ggcagtggcc agatactgcg acctccctag caaactgggg cacaagctta attgagcggc 2341 cgctcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc 2401 tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg 2461 tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt 2521 gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac 2581 tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc 2641 tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct 2701 gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct 2761 cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct 2821 caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct 2881 acgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc aggtaccttt 2941 aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag aaaagggggg 3001 actggaaggg ctaattcact cccaaagaag tcaagatctg ctttttgcct gtactgggtc 3061 tctctggtta gaccagagtc tctctggtta gaccagatct gagcctggga gctctctggc 3121 taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg 3181 tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg 3241 tggaaaatct ctagcagttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca 3301 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac 3361 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat 3421 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagtca atagcaggca 3481 tgctggggat gcggtgggct ctatgggcgg ccgttaatta agcttttgtt ccctttagtg 3541 agggttaatt gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta 3601 tccgctcaca attccacaca acatacgagc cgggagcata aagtgtaaag cctggggtgc 3661 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg 3721 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg 3781 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg 3841 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa 3901 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc 3961 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc 4021 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag 4081 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct 4141 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta 4201 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc 4261 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc 4321 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt 4381 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct 4441 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc 4501 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca 4561 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta 4621 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa 4681 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg 4741 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg 4801 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc 4861 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc 4921 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa 4981 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc 5041 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg 5101 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc 5161 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat 5221 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg 5281 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc 5341 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg 5401 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat 5461 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg 5521 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg 5581 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct 5641 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac 5701 atttccccga aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta 5761 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat 5821 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca 5881 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc 5941 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta 6001 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg 6061 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg 6121 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc 6181 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta 6241 ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg 6301 ttttcccagt cacgacgttg taaaacgacg gccagtgagc gcgcgtaata cgactcacta 6361 tagggcgaat tgactagtac ataacttacg gtaaatggcc cgcctggctg accgcccaac 6421 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact 6481 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa 6541 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg 6601 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta 6661 gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg 6721 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg 6781 caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg 6841 ggcggtaggc gtgtacggtg ggaggtctat ataagcagcg cgttttgcct gtactgggtc 6901 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct 6961 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga 7021 ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg 7081 cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact 7141 cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa 7201 attttgacta gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg 7261 gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat 7321 aaattaaaac atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc 7381 ctgttagaaa catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag 7441 acaggatcag aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat 7501 caaaggatag agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac 7561 aaaagtaaga ccaccgcaca gcaagcggcc ggccgcgctg atcttcagac ctggaggagg 7621 agatatgagg gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc 7681 attaggagta gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt 7741 gggaatagga gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc 7801 gtcaatgacg ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa 7861 caatttgctg agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat 7921 caagcagctc caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct 7981 ggggatttgg ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag 8041 ttggagtaat aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag 8101 agaaattaac aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca 8161 agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt 8221 taacataaca aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt 8281 aggtttaaga atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc 8341 accattatcg tttcagaccc acctcccaac cccgagggga cccgacaggc ccgaaggaat 8401 agaagaagaa ggtggagaga gagacagaga cagatccatt cgattagtga acggatcggc 8461 actgcgtgcg ccaattctgc agacaaatgg cagtattcat ccacaatttt aaaagaaaag 8521 gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac 8581 aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg 8641 acagcagaga tccagtttgg ttagtaccgg gccctacgcg tccaaggtcg ggcaggaa //