LOCUS Exported 7324 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION CRISPR-Sirius plasmid. ACCESSION . VERSION . KEYWORDS pHAGE-EFS-PCP-GFPnls SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7324) AUTHORS Ma H, Tu LC, Naseri A, Chung YC, Grunwald D, Zhang S, Pederson T TITLE CRISPR-Sirius: RNA scaffolds for signal amplification in genome imaging. JOURNAL Nat Methods. 2018 Nov;15(11):928-931. doi: 10.1038/s41592-018-0174-0. Epub 2018 Oct 30. PUBMED 30377374 REFERENCE 2 (bases 1 to 7324) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7324 /organism="synthetic DNA construct" /mol_type="other DNA" LTR 1..634 /label=3' LTR /note="3' long terminal repeat (LTR) from HIV-1" misc_feature 681..806 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1303..1536 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 1721..1765 /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 2058..2175 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 2265..2476 /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" regulatory 2522..2531 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2933..3646 /codon_start=1 /product="GFP variant that folds robustly even when fused to poorly folded proteins (Pedelacq et al., 2006)" /label=superfolder GFP /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE FVTAAGITHGMDELYK" CDS 3683..3703 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" misc_feature 3714..4302 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(3767..3787) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(4185..4196) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 4377..4610 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" primer_bind complement(4729..4747) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 4815..4919 /gene="bla" /label=AmpR promoter CDS 4920..5780 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(5138..5157) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5951..6539 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6440..6459 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 6693..6710 /label=L4440 /note="L4440 vector, forward primer" primer_bind 6802..6821 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" protein_bind 6950..6971 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 6986..7016 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 7024..7040 /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 7029..7051 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 7048..7064 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 7048..7064 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 7114..7133 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" ORIGIN 1 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca 61 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac 121 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca 181 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg 241 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag 301 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg 361 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat 421 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga 481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct 541 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc 601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag 661 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg 721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga 781 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg 841 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg 901 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct 961 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat 1021 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca 1081 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc 1141 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag 1201 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc 1261 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg 1321 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc 1381 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc 1441 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct 1501 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa 1561 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca 1621 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt 1681 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt 1741 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta 1801 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt 1861 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct 1921 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga 1981 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccgaa ttgacaaatg 2041 gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa 2101 agaatagtag acataatagc aacagacata caaactaaag aattacaaaa acaaattaca 2161 aaaattcaaa attttcgggt ttattacagg gacagcagag atccagtttg gactagcgat 2221 gctagctgat gcgggccctt cgagtggctc cggtgcccgt cagtgggcag agcgcacatc 2281 gcccacagtc cccgagaagt tggggggagg ggtcggcaat tgaaccggtg cctagagaag 2341 gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg 2401 tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt 2461 tgccgccaga acacaggtgt cgtgacgcgg ttccggttaa ggtgaacgcg tgcaggctgg 2521 cgccaccatg ggttccaaaa ccatcgttct ttcggtcggc gaggctactc gcactctgac 2581 tgagatccag tccaccgcag accgtcagat cttcgaagag aaggtcgggc ctctggtggg 2641 tcggctgcgc ctcacggctt cgctccgtca aaacggagcc aagaccgcgt atcgcgtcaa 2701 cctaaaactg gatcaggcgg acgtcgttga ttccggactt ccgaaagtgc gctacactca 2761 ggtatggtcg cacgacgtga caatcgttgc gaatagcacc gaggcctcgc gcaaatcgtt 2821 gtacgatttg accaagtccc tcgtcgcgac ctcgcaggtc gaagatcttg tcgtcaacct 2881 tgtgccgctg ggccgtggtg gcggagggac tagtggaggt ggatccggaa gtatgcgtaa 2941 aggcgaagag ctgttcactg gtgtcgtccc tattctggtg gaactggatg gtgatgtcaa 3001 cggtcataag ttttccgtgc gtggcgaggg tgaaggtgac gcaactaatg gtaaactgac 3061 gctgaagttc atctgtacta ctggtaaact gccggtacct tggccgactc tggtaacgac 3121 gctgacttat ggtgttcagt gctttgctcg ttatccggac catatgaagc agcatgactt 3181 cttcaagtcc gccatgccgg aaggctatgt gcaggaacgc acgatttcct ttaaggatga 3241 cggcacgtac aaaacgcgtg cggaagtgaa atttgaaggc gataccctgg taaaccgcat 3301 tgagctgaaa ggcattgact ttaaagaaga cggcaatatc ctgggccata agctggaata 3361 caattttaac agccacaatg tttacatcac cgccgataaa caaaaaaatg gcattaaagc 3421 gaattttaaa attcgccaca acgtggagga tggcagcgtg cagctggctg atcactacca 3481 gcaaaacact ccaatcggtg atggtcctgt tctgctgcca gacaatcact atctgagcac 3541 gcaaagcgtt ctgtctaaag atccgaacga gaaacgcgat catatggttc tgctggagtt 3601 cgtaaccgca gcgggcatca cgcatggtat ggatgaactg tacaaatctg gcggccgctc 3661 tggcatcgat gggagcctcg agccaaagaa aaagcggaaa gtgtaatcta gataatcaac 3721 ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta 3781 cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt 3841 tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg 3901 ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg 3961 gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca 4021 cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca 4081 ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctgtg 4141 ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag 4201 cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc 4261 gccctcagac gagtcggatc tccctttggg ccgcctcccc gcctgagatc ctttaagacc 4321 aatgacttac aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga 4381 agggctaatt cactcccaac gaagacaaga tctgcttttt gcttgtactg ggtctctctg 4441 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc 4501 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg 4561 taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtagtagttc 4621 atgtcatctt attattcagt atttataact tgcaaagaaa tgaatatcag agagtgagag 4681 gcccgggtta attaaggaaa gggctagatc attcttgaag acgaaagggc ctcgtgatac 4741 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt 4801 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt 4861 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta 4921 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg 4981 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac 5041 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg 5101 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc 5161 gtgttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg 5221 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat 5281 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg 5341 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg 5401 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc 5461 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt 5521 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct 5581 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc 5641 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca 5701 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct 5761 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt 5821 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga 5881 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca 5941 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac 6001 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg 6061 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag 6121 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac 6181 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt 6241 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg 6301 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc 6361 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc 6421 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc 6481 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa 6541 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt 6601 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg 6661 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag 6721 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagcaagc 6781 tcatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct 6841 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctccccgt 6901 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt 6961 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg 7021 gaattgtgag cggataacaa tttcacacag gaaacagcta tgacatgatt acgaatttca 7081 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat 7141 cttatcatgt ctggatcaac tggataactc aagctaacca aaatcatccc aaacttccca 7201 ccccataccc tattaccact gccaattacc tgtggtttca tttactctaa acctgtgatt 7261 cctctgaatt attttcattt taaagaaatt gtatttgtta aatatgtact acaaacttag 7321 tagt //