LOCUS Exported 8308 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Photoactivatable transcription system. Lentiviral expression of IL1RN sgRNA3. Also contains a CMV-puro-t2A-mCherry expression cassette. ACCESSION . VERSION . KEYWORDS sgRNA3_IL1RN SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8308) AUTHORS Nihongaki Y, Yamamoto S, Kawano F, Suzuki H, Sato M TITLE CRISPR-Cas9-based Photoactivatable Transcription System. JOURNAL Chem Biol. 2015 Feb 19;22(2):169-74. doi: 10.1016/j.chembiol.2014.12.011. Epub 2015 Jan 22. PUBMED 25619936 REFERENCE 2 (bases 1 to 8308) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8308 /organism="synthetic DNA construct" /mol_type="other DNA" protein_bind 209..242 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind 246..266 /label=mU6-F /note="Mouse U6 promoter, forward primer" misc_RNA 349..424 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" enhancer 549..852 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 853..1056 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" CDS 1090..1686 /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(1090..1109) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 1586..1606 /label=Puro-F /note="Puromycin resistance gene, forward primer" CDS 1696..1749 /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" regulatory 1756..1765 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1762..2472 /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA EGRHSTGGMDELYK" primer_bind complement(1907..1925) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 2175..2194 /label=mCherry-F /note="mCherry, forward primer" protein_bind 2492..2525 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 2581..3169 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(2634..2654) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3052..3063) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(3172..3188) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3173..3189) /label=pBluescriptKS /note="For pBluescript vector" LTR 3698..3878 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" rep_origin complement(3940..4528) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4020..4039) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4699..5559) /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 5322..5341 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5560..5664) /gene="bla" /label=AmpR promoter primer_bind complement(5748..5767) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 5939..6318 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 6320..6518 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" LTR 6536..6716 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 6763..6888 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 7387..7620 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 7805..7849 /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 8147..8264 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" ORIGIN 1 gggcccgctc tagagatccg acgcgccatc tctaggcccg cgccggcccc ctcgcacaga 61 cttgtgggag aagctcggct actcccctgc cccggttaat ttgcatataa tatttcctag 121 taactataga ggcttaatgt gcgataaaag acagataatc tgttcttttt aatactagct 181 acattttaca tgataggctt ggatttctat aacttcgtat agcatacatt atacgaagtt 241 ataaacagca caaaaggaaa ctcaccctaa ctgtaaagta attgtgtgtt ttgagactat 301 aagtatccct tggagaacca ccttgttggc atcaagtcag ccatcagcgt tttagagcta 361 gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg 421 gtgctttttt tctcgagtac taggatccat taggcggccg cgtggataac cgtattaccg 481 ccatgcatta gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg 541 gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc 601 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat 661 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat 721 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat 781 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc 841 gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac 901 tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa 961 aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt 1021 aggcgtgtac ggtgggaggt ctatataagc agagctggtt tagtgaaccg tcagatccgc 1081 tagcccacca tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc 1141 cgggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc 1201 gacccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc 1261 gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc 1321 acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag 1381 ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg 1441 cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca ccagggcaag 1501 ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc 1561 gccttcctgg agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc 1621 gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc 1681 ggtgccggat cgggagaggg cagaggaagt ctgctaacat gcggtgacgt cgaggagaat 1741 cctggcccac cggtcgccac catggtgagc aagggcgagg aggataacat ggccatcatc 1801 aaggagttca tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca cgagttcgag 1861 atcgagggcg agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg 1921 accaagggtg gccccctgcc cttcgcctgg gacatcctgt cccctcagtt catgtacggc 1981 tccaaggcct acgtgaagca ccccgccgac atccccgact acttgaagct gtccttcccc 2041 gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc 2101 caggactcct ccctgcagga cggcgagttc atctacaagg tgaagctgcg cggcaccaac 2161 ttcccctccg acggccccgt aatgcagaag aagaccatgg gctgggaggc ctcctccgag 2221 cggatgtacc ccgaggacgg cgccctgaag ggcgagatca agcagaggct gaagctgaag 2281 gacggcggcc actacgacgc tgaggtcaag accacctaca aggccaagaa gcccgtgcag 2341 ctgcccggcg cctacaacgt caacatcaag ttggacatca cctcccacaa cgaggactac 2401 accatcgtgg aacagtacga acgcgccgag ggccgccact ccaccggcgg catggacgag 2461 ctgtacaagt aagaattcgt cgagggacct aataacttcg tatagcatac attatacgaa 2521 gttatacatg tttaagggtt ccggttccac taggtacaat tcgatatcaa gcttatcgat 2581 aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct 2641 ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt 2701 atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg 2761 tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact 2821 ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct 2881 attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg 2941 ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc 3001 gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc 3061 aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt 3121 cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgca tcgataccgt 3181 cgacctcgat cgagacctag aaaaacatgg agcaatcaca agtagcaata cagcagctac 3241 caatgctgat tgtgcctggc tagaagcaca agaggaggag gaggtgggtt ttccagtcac 3301 acctcaggta cctttaagac caatgactta caaggcagct gtagatctta gccacttttt 3361 aaaagaaaag gggggactgg aagggctaat tcactcccaa cgaagacaag atatccttga 3421 tctgtggatc taccacacac aaggctactt ccctgattgg cagaactaca caccagggcc 3481 agggatcaga tatccactga cctttggatg gtgctacaag ctagtaccag ttgagcaaga 3541 gaaggtagaa gaagccaatg aaggagagaa cacccgcttg ttacaccctg tgagcctgca 3601 tgggatggat gacccggaga gagaagtatt agagtggagg tttgacagcc gcctagcatt 3661 tcatcacatg gcccgagagc tgcatccgga ctgtactggg tctctctggt tagaccagat 3721 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt 3781 gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc 3841 cctcagaccc ttttagtcag tgtggaaaat ctctagcagc atgtgagcaa aaggccagca 3901 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc 3961 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 4021 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 4081 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc 4141 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 4201 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 4261 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 4321 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag 4381 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 4441 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 4501 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 4561 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat 4621 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga 4681 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg 4741 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga 4801 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc 4861 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac 4921 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc 4981 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc 5041 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc 5101 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt 5161 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc 5221 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg 5281 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag 5341 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat 5401 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc 5461 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa 5521 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta 5581 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa 5641 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgcttaagc 5701 ggtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct 5761 gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag 5821 tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat 5881 ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga 5941 cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca 6001 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac 6061 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact 6121 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa 6181 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg 6241 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta 6301 gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg 6361 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg 6421 caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg 6481 ggcggtaggc gtgtacggtg ggaggtctat ataagcagcg cgttttgcct gtactgggtc 6541 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct 6601 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga 6661 ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg 6721 cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact 6781 cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa 6841 attttgacta gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg 6901 gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat 6961 aaattaaaac atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc 7021 ctgttagaaa catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag 7081 acaggatcag aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat 7141 caaaggatag agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac 7201 aaaagtaaga ccaccgcaca gcaagcggcc ggccgcgctg atcttcagac ctggaggagg 7261 agatatgagg gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc 7321 attaggagta gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt 7381 gggaatagga gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc 7441 gtcaatgacg ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa 7501 caatttgctg agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat 7561 caagcagctc caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct 7621 ggggatttgg ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag 7681 ttggagtaat aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag 7741 agaaattaac aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca 7801 agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt 7861 taacataaca aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt 7921 aggtttaaga atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc 7981 accattatcg tttcagaccc acctcccaac cccgagggga cccgacaggc ccgaaggaat 8041 agaagaagaa ggtggagaga gagacagaga cagatccatt cgattagtga acggatcggc 8101 actgcgtgcg ccaattctgc agacaaatgg cagtattcat ccacaatttt aaaagaaaag 8161 gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac 8221 aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg 8281 acagcagaga tccagtttgg ttagtacc //