LOCUS Exported 8694 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION for CRISPRa, lentiviral expression of gRNA scaffold using activator P65 HSF. ACCESSION . VERSION . KEYWORDS pXPR_502 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8694) 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 8694) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8694 /organism="synthetic DNA construct" /mol_type="other DNA" LTR 104..337 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" CDS complement(427..1023) /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(507..527) /label=Puro-F /note="Puromycin resistance gene, forward primer" primer_bind 1004..1023 /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" CDS complement(1051..1104) /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 complement(1120..1491) /codon_start=1 /product="C-terminal activation domain from the human heat shock transcription factor HSF1" /label=HSF1 /translation="GFSVDTSALLDLFSPSVTVPDMSLPDLDSSLASIQELLSPQEPPR PPEAENSSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPVLFELGEGSYFSEGDGFAED PTISLLTGSEPPKAKDPTVS" CDS complement(1516..2058) /codon_start=1 /product="C-terminal portion of the p65 subunit of mouse NF-kappa-B" /label=p65 /translation="PSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTP GPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSE FQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDED FSSIADMDFSALLSQISS" CDS complement(2074..2094) /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" protein_bind 2144..2168 /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" promoter complement(2633..3133) /label=hPGK promoter /note="human phosphoglycerate kinase 1 promoter" primer_bind complement(2696..2714) /label=hPGK-F /note="Human PGK promoter, forward primer" misc_feature 3192..3309 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter complement(3591..3831) /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind complement(3641..3660) /label=LKO.1 5' /note="Human U6 promoter, forward primer" primer_bind complement(3811..3831) /label=hU6-F /note="Human U6 promoter, forward primer" CDS complement(4009..4053) /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 4238..4471 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." misc_feature 4964..5089 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" LTR 5136..5316 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" promoter complement(5317..5543) /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" primer_bind complement(5571..5591) /label=T3 /note="T3 promoter, forward primer" promoter complement(5571..5589) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(5610..5626) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5610..5626) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5623..5645) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5634..5650 /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(5658..5688) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5703..5724 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5841..5858) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6012..6600) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6092..6111) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6771..7631) /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 7394..7413 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7632..7736) /gene="bla" /label=AmpR promoter rep_origin complement(7762..8217) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(7899..7920) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 8111..8130 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 8344..8366 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 8358..8375 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 8359..8375 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 8385..8404 /label=T7 /note="T7 promoter, forward primer" promoter 8385..8403 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" rep_origin 8424..8559 /label=SV40 ori /note="SV40 origin of replication" primer_bind complement(8478..8497) /label=SV40pro-F /note="SV40 promoter/origin, forward primer" polyA_signal 8599..26 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(8610..8629) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 8664..8683 /label=SV40pA-R /note="SV40 polyA, reverse primer" ORIGIN 1 ccattataag ctgcaataaa caagttcctc tcactctctg atattcattt ctttgcaagt 61 tataaatact gaataataag atgacatgaa ctactatacg tactgctaga gattttccac 121 actgactaaa agggtctgag ggatctctag ttaccagagt cacacaacag acgggcacac 181 actacttgaa gcactcaagg caagctttat tgaggcttaa gcagtgggtt ccctagttag 241 ccagagagct cccaggctca gatctggtct aaccagagag acccagtaca agcaaaaagc 301 agatcttgtc ttcgttggga gtgaattagc ccttccagtc cccccttttc ttttaaaaag 361 tggctaagat ctacagctgc cttgtaagtc attggtctta aagtcgactt aacgcgttca 421 gctagcggca ccgggcttgc gggtcatgca ccaggtgcgc ggtccttcgg gcacctcgac 481 gtcggcggtg acggtgaagc cgagccgctc gtagaagggg aggttgcggg gcgcggaggt 541 ctccaggaag gcgggcaccc cggcgcgctc ggccgcctcc actccgggga gcacgacggc 601 gctgcccaga cccttgccct ggtggtcggg cgaaacgccg acggtggcca ggaaccacgc 661 gggctccttg ggccggtgcg gcgccaggag gccttccatc tgttgctgcg cggccagccg 721 ggaaccgctc aactcggcca tgcgcgggcc gatctcggcg aacaccgccc ccgcttcgac 781 gctctccggc gtggtccaga ccgccacggc ggcgccgtcg tccgcgaccc acaccttgcc 841 gatgtcgagc ccgacgcgcg tgaggaagag ttcttgcagc tcggtgaccc gctcgatgtg 901 gcggtccgga tcgacggtgt ggcgcgtggc ggggtagtcg gcgaacgcgg cggcgagggt 961 gcgtacggcc ctggggacgt cgtcgcgggt ggcgaggcgc accgtgggct tgtactcggt 1021 catggtaagc tccggtggat ctgtggtacc tgggccagga ttctcctcga cgtcaccgca 1081 tgttagcaga cttcctctgc cctctccact gccgatatcg gagacagtgg ggtccttggc 1141 tttgggaggc tccgagcctg tcagcaggga gatggtgggg tcctcggcga agccgtcccc 1201 ttcggagaag taggagccct ctcccagctc aaacagcacc ggcaggtcgt tgctcccggt 1261 gtccacggag ccggggtcca gcaggaacag cggctgcgct gtgtagtgca ccagctgctt 1321 ccctgaatcc gggctgctgt tctctgcctc gggaggcctg gggggctcct ggggagacag 1381 gagctcttgg atactggcca ggctgctgtc aaggtcaggc aggctcatgt cgggcacggt 1441 caccgagggg ctgaacaggt ccagcagggc actggtgtcc acgctgaagc cgcttccacc 1501 tcctccctgc ccactagagg aaatctgtga cagcagggca ctaaagtcca tatcagcgat 1561 gcttgagaag tcttcatctc cggacagccc attaggcagg ccgctggttc ccaggggagt 1621 tggagcgggg tcggggggcc gctggctgcc ggtcaccagc cgggtaatgg cttcggggta 1681 ctccatcagc attggttcgg ctgtactatg agacatggac acgccctgat tcagcagctg 1741 ctgaaactca gagttgtcca cggaggccag atctgtgaac actccgggat cggtgctgtt 1801 ccccagcaga gctcccaggt cctcatcagc gtcgaactgc aggtgcagca gagcttcact 1861 cagagtcccc tcgccggcct gtgtagactt gggcactgga gcgctcagtg actggggtgg 1921 tcctggggtc agcacagggg ctggagcagg tggctgggcc agaggcacca tagcactaga 1981 gggcaccata gtctgggcca gcactggagc ggagctaggg gccagagcca gggcctggtt 2041 gctgatctgc cctgaagggg taccagcggc cgccaccttc ctctttttct taggtccgct 2101 acctcctcct ccgcttcctc ctcctccgct tccacctcct ccgcccactt tgtacaagaa 2161 agttgaagaa cggcccagcg gcacaaggtt gacgacaaga tcttcgacct gcgaggtcgc 2221 gacgagggac ttggtcaaat cgtacaacga tttgcgcgag gcctcggtgc tattcgcaac 2281 gattgtcacg tcgtgcgacc atacctgagt gtagcgcact ttcggaagtc cggaatcaac 2341 gacgtccgcc tgatccagtt ttaggttgac tcgatacgcg gtcttggctc cgttttgacg 2401 gagcgaagcc gtgaggcgca gccgacccac cagaggcccg accttctctt cgaagatctg 2461 acggtctgcg gtggactgga tctcagtcag agtgcgagta gcctcgccga ccgaaagaac 2521 gatggttttg gaggctagca tagacgtctt cctccttttg tgaggttgtc caccatcgtt 2581 ctcgtagctg tgccgcctcc gcgacatggt ggcaagctta agttggatcc ccctggggag 2641 agaggtcggt gattcggtca acgagggagc cgactgccga cgtgcgctcc ggaggcttgc 2701 agaatgcgga acaccgcgcg ggcaggaaca gggcccacac taccgcccca caccccgcct 2761 cccgcaccgc cccttcccgg ccgctgctct cggcgcgccc tgctgagcag ccgctattgg 2821 ccacagccca tcgcggtcgg cgcgctgcca ttgctccctg gcgctgtccg tctgcgaggg 2881 tactagtaag acgtgcggct tccgtttgtc acgtccggca cgccgcgaac cgcaaggaac 2941 cttcccgact taggggcgga gcaggaagcg tcgccggggg gcccacaagg gtagcggcga 3001 agatccgggt gacgctgcga acggacgtga agaatgtgcg agacccaggg tcggcgccgc 3061 tgcgtttccc ggaaccacgc ccagagcagc cgcgtccctg cgcaaaccca gggctgcctt 3121 ggaaaaggcg caacccgggc cccctcgagc cggcgccaaa gtggatctct gctgtccctg 3181 taataaaccc gaaaattttg aatttttgta atttgttttt gtaattcttt agtttgtatg 3241 tctgttgcta ttatgtctac tattctttcc cctgcactgt accccccaat cccccctttt 3301 cttttaaaat tgtggatgaa tactgccatt tgtctcaaga tctagaattc aaaaaaagca 3361 ccgactcggt gccacttttt caagttgata acggactagc cttatttaaa cttgctatgc 3421 tgcgaatacg agcagaccct gatggtgtct gatacgtacc ctgatggtgt acgagaccac 3481 ggagcccata tgctctcgtg gagaggagcg acgccatatc gtctgctccc tcgtattcgc 3541 agcatagctc ttaaacagag acgcaaaaaa ccggtcgtct ccggtgtttc gtcctttcca 3601 caagatatat aaagccaaga aatcgaaata ctttcaagtt acggtaagca tatgatagtc 3661 cattttaaaa cataatttta aaactgcaaa ctacccaaga aattattact ttctacgtca 3721 cgtattttgt actaatatct ttgtgtttac agtcaaatta attctaatta tctctctaac 3781 agccttgtat cgtatatgca aatatgaagg aatcatggga aataggccct ctctgggtcc 3841 cctcggggtt gggaggtggg tctgaaacga taatggtgaa tatccctgcc taactctatt 3901 cactatagaa agtacagcaa aaactattct taaacctacc aagcctccta ctatcattat 3961 gaataatttt atataccaca gccaatttgt tatgttaaac caattccaca aacttgccca 4021 tttatctaat tccaataatt cttgttcatt cttttcttgc tggttttgcg attcttcaat 4081 taaggagtgt attaagcttg tgtaattgtt aatttctctg tcccactcca tccaggtcgt 4141 gtgattccaa atctgttcca gagatttatt actccaacta gcattccaag gcacagcagt 4201 ggtgcaaatg agttttccag agcaacccca aatccccagg agctgttgat cctttaggta 4261 tctttccaca gccaggattc ttgcctggag ctgcttgatg ccccagactg tgagttgcaa 4321 cagatgctgt tgcgcctcaa tagccctcag caaattgttc tgctgctgca ctataccaga 4381 caataattgt ctggcctgta ccgtcagcgt cattgacgct gcgcccatag tgcttcctgc 4441 tgctcccaag aacccaagga acaaagctcc tattcccact gctctttttt ctctctgcac 4501 cactcttctc tttgccttgg tgggtgctac tcctaatggt tcaattttta ctactttata 4561 tttatataat tcacttctcc aattgtccct catatctcct cctccaggtc tgaagatcag 4621 cggccgcttg ctgtgcggtg gtcttacttt tgttttgctc ttcctctatc ttgtctaaag 4681 cttccttggt gtcttttatc tctatccttt gatgcacaca atagagggtt gctactgtat 4741 tatataatga tctaagttct tctgatcctg tctgaaggga tggttgtagc tgtcccagta 4801 tttgtctaca gccttctgat gtttctaaca ggccaggatt aactgcgaat cgttctagct 4861 ccctgcttgc ccatactata tgttttaatt tatatttttt ctttccccct ggccttaacc 4921 gaattttttc ccatcgcgat ctaattctcc cccgcttaat actgacgctc tcgcacccat 4981 ctctctcctt ctagcctccg ctagtcaaaa tttttggcgt actcaccagt cgccgcccct 5041 cgcctcttgc cgtgcgcgct tcagcaagcc gagtcctgcg tcgagagagc tcctctggtt 5101 tccctttcgc tttcaagtcc ctgttcgggc gccactgcta gagattttcc acactgacta 5161 aaagggtctg agggatctct agttaccaga gtcacacaac agacgggcac acactacttg 5221 aagcactcaa ggcaagcttt attgaggctt aagcagtggg ttccctagtt agccagagag 5281 ctcccaggct cagatctggt ctaaccagag agacccgttt atgtatcgag ctaggcactt 5341 aaatacaata tctctgcaat gcggcaattc agtggttcgt ccaatccatg tcagacccgt 5401 ctgttgcctt cctaataagg cacgatcgta ccaccttact tccaccaatc ggcatgcacg 5461 gtgctttttc tctccttgta aggcatgttg ctaactcatc gttaccatgt tgcaagacta 5521 caagagtatt gcataagact acattaagct tgcagctcca gcttttgttc cctttagtga 5581 gggttaattg cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat 5641 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc 5701 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga 5761 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt 5821 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 5881 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 5941 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 6001 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 6061 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 6121 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 6181 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag 6241 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 6301 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 6361 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 6421 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg 6481 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 6541 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 6601 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 6661 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa 6721 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc 6781 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga 6841 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca 6901 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc 6961 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat 7021 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc 7081 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt 7141 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc 7201 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg 7261 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt 7321 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg 7381 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga 7441 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg 7501 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg 7561 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt 7621 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc 7681 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca 7741 tttccccgaa aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa 7801 atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata 7861 aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac 7921 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc 7981 cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa 8041 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg 8101 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg 8161 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc 8221 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 8281 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 8341 tttcccagtc acgacgttgt aaaacgacgg ccagtgagcg cgcgtaatac gactcactat 8401 agggcgaatt gggtacgtcc ctaggcctcc aaaaaagcct cctcactact tctggaatag 8461 ctcagaggcc gaggcggcct cggcctctgc ataaataaaa aaaattagtc agccatgggg 8521 cggagaatgg gcggaactgg gcggagttag gggcgggatg ggcggagtta ggggcgggat 8581 agctagagcc agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc 8641 agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaa //