LOCUS Exported 7920 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION for CRISPRa, lentiviral expression of gRNA scaffold using activator VP64. ACCESSION . VERSION . KEYWORDS pXPR_501 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7920) AUTHORS Sanson KR, Hanna RE, Hegde M, Donovan KF, Strand C, Sullender ME, Vaimberg EW, Goodale A, Root DE, Piccioni F, Doench JG TITLE Optimized libraries for CRISPR-Cas9 genetic screens with multiple modalities. JOURNAL Nat Commun. 2018 Dec 21;9(1):5416. doi: 10.1038/s41467-018-07901-8. PUBMED 30575746 REFERENCE 2 (bases 1 to 7920) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7920 /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 1717..1957 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 1717..1737 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 1888..1907 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_feature 2239..2356 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 2415..2915 /label=hPGK promoter /note="human phosphoglycerate kinase 1 promoter" primer_bind 2834..2852 /label=hPGK-F /note="Human PGK promoter, forward primer" protein_bind complement(3380..3404) /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" CDS 3454..3474 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 3499..3648 /codon_start=1 /product="tetrameric repeat of the minimal activation domain of herpes simplex virus VP16 (Beerli et al., 1998)" /label=VP64 /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF DLDML" CDS 3670..3723 /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 3751..4347 /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(3751..3770) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 4247..4267 /label=Puro-F /note="Puromycin resistance gene, forward primer" LTR 4437..4670 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 4748..4869 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4785..4804) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4839..4858 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 4909..5044 /label=SV40 ori /note="SV40 origin of replication" primer_bind 4971..4990 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(5064..5083) /label=T7 /note="T7 promoter, forward primer" promoter complement(5065..5083) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(5093..5110) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(5093..5109) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5102..5124) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 5251..5706 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5338..5357) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5548..5569 /label=F1ori-F /note="F1 origin, forward primer" promoter 5732..5836 /gene="bla" /label=AmpR promoter CDS 5837..6697 /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(6055..6074) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6868..7456 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7357..7376 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 7610..7627 /label=L4440 /note="L4440 vector, forward primer" protein_bind 7744..7765 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 7780..7810 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 7818..7834 /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 7823..7845 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 7842..7858 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 7842..7858 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 7877..7897 /label=T3 /note="T3 promoter, forward primer" promoter 7879..7897 /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 cccagagagg gcctatttcc catgattcct 1741 tcatatttgc atatacgata caaggctgtt agagagataa ttagaattaa tttgactgta 1801 aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt 1861 gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat 1921 ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccggaga cgaccggttt 1981 tttgcgtctc tgtttaagag ctatgctgcg aatacgaggg agcagacgat atggcgtcgc 2041 tcctctccac gagagcatat gggctccgtg gtctcgtaca ccatcagggt acgtatcaga 2101 caccatcagg gtctgctcgt attcgcagca tagcaagttt aaataaggct agtccgttat 2161 caacttgaaa aagtggcacc gagtcggtgc tttttttgaa ttctagatct tgagacaaat 2221 ggcagtattc atccacaatt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga 2281 aagaatagta gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac 2341 aaaaattcaa aattttcggg tttattacag ggacagcaga gatccacttt ggcgccggct 2401 cgagggggcc cgggttgcgc cttttccaag gcagccctgg gtttgcgcag ggacgcggct 2461 gctctgggcg tggttccggg aaacgcagcg gcgccgaccc tgggtctcgc acattcttca 2521 cgtccgttcg cagcgtcacc cggatcttcg ccgctaccct tgtgggcccc ccggcgacgc 2581 ttcctgctcc gcccctaagt cgggaaggtt ccttgcggtt cgcggcgtgc cggacgtgac 2641 aaacggaagc cgcacgtctt actagtaccc tcgcagacgg acagcgccag ggagcaatgg 2701 cagcgcgccg accgcgatgg gctgtggcca atagcggctg ctcagcaggg cgcgccgaga 2761 gcagcggccg ggaaggggcg gtgcgggagg cggggtgtgg ggcggtagtg tgggccctgt 2821 tcctgcccgc gcggtgttcc gcattctgca agcctccgga gcgcacgtcg gcagtcggct 2881 ccctcgttga ccgaatcacc gacctctctc cccaggggga tccaacttaa gcttgccacc 2941 atgtcgcgga ggcggcacag ctacgagaac gatggtggac aacctcacaa aaggaggaag 3001 acgtctatgc tagcctccaa aaccatcgtt ctttcggtcg gcgaggctac tcgcactctg 3061 actgagatcc agtccaccgc agaccgtcag atcttcgaag agaaggtcgg gcctctggtg 3121 ggtcggctgc gcctcacggc ttcgctccgt caaaacggag ccaagaccgc gtatcgagtc 3181 aacctaaaac tggatcaggc ggacgtcgtt gattccggac ttccgaaagt gcgctacact 3241 caggtatggt cgcacgacgt gacaatcgtt gcgaatagca ccgaggcctc gcgcaaatcg 3301 ttgtacgatt tgaccaagtc cctcgtcgcg acctcgcagg tcgaagatct tgtcgtcaac 3361 cttgtgccgc tgggccgttc ttcaactttc ttgtacaaag tgggcggagg aggtggaagc 3421 ggaggaggag gaagcggagg aggaggtagc ggacctaaga aaaagaggaa ggtggcggcc 3481 gctggtaccg gacgggctga cgcattggac gattttgatc tggatatgct gggaagtgac 3541 gccctcgatg attttgacct tgacatgctt ggttcggatg cccttgatga ctttgacctc 3601 gacatgctcg gcagtgacgc ccttgatgat ttcgacctgg acatgctgat taacgatatc 3661 ggcagtggag agggcagagg aagtctgcta acatgcggtg acgtcgagga gaatcctggc 3721 ccaggtacca cagatccacc ggagcttacc atgaccgagt acaagcccac ggtgcgcctc 3781 gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac 3841 cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa 3901 gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc 3961 gccgccgtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag 4021 atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa 4081 ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtt 4141 tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg 4201 gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc 4261 tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc 4321 tggtgcatga cccgcaagcc cggtgccgct agctgaacgc gttaagtcga ctttaagacc 4381 aatgacttac aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga 4441 agggctaatt cactcccaac gaagacaaga tctgcttttt gcttgtactg ggtctctctg 4501 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc 4561 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg 4621 taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtacgtatag 4681 tagttcatgt catcttatta ttcagtattt ataacttgca aagaaatgaa tatcagagag 4741 tgagaggaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa 4801 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa 4861 tgtatcttat catgtctggc tctagctatc ccgcccctaa ctccgcccat cccgccccta 4921 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca 4981 gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga 5041 ggcctaggga cgtacccaat tcgccctata gtgagtcgta ttacgcgcgc tcactggccg 5101 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag 5161 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc 5221 aacagttgcg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg 5281 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc 5341 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa 5401 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac 5461 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt 5521 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca 5581 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt 5641 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta 5701 caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta 5761 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata 5821 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc 5881 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga 5941 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct 6001 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg 6061 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta 6121 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat 6181 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt 6241 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga 6301 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga 6361 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga 6421 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc 6481 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc 6541 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg 6601 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat 6661 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata 6721 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct 6781 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga 6841 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg 6901 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc 6961 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct 7021 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc 7081 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt 7141 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg 7201 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct 7261 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag 7321 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag 7381 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg 7441 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg 7501 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac 7561 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt 7621 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat 7681 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc 7741 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc 7801 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca 7861 tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg agctgcaagc //