LOCUS Exported 8557 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION CRISPR-Sirius plasmid. ACCESSION . VERSION . KEYWORDS pLH-sgRNA-Sirius-8XMS2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8557) 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 8557) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8557 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 426..444 /label=hPGK-F /note="Human PGK promoter, forward primer" CDS 597..1622 /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /label=HygR /note="confers resistance to hygromycin" /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK E" primer_bind complement(1646..1662) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(1647..1663) /label=pBluescriptKS /note="For pBluescript vector" LTR 1745..1978 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 2050..2171 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2087..2106) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 2141..2160 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 2211..2346 /label=SV40 ori /note="SV40 origin of replication" primer_bind 2273..2292 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(2366..2385) /label=T7 /note="T7 promoter, forward primer" promoter complement(2367..2385) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(2395..2412) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(2395..2411) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2404..2426) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 2553..3008 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2640..2659) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2850..2871 /label=F1ori-F /note="F1 origin, forward primer" promoter 3034..3138 /gene="bla" /label=AmpR promoter CDS 3139..3999 /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(3357..3376) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 4170..4758 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4659..4678 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4912..4929 /label=L4440 /note="L4440 vector, forward primer" protein_bind 5046..5067 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 5082..5112 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5120..5136 /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 5125..5147 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 5144..5160 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 5144..5160 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 5179..5199 /label=T3 /note="T3 promoter, forward primer" promoter 5181..5199 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 5227..5453 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 5454..5634 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 5681..5806 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 6299..6532 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 6717..6761 /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 7059..7299 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 7059..7079 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 7230..7249 /label=LKO.1 5' /note="Human U6 promoter, forward primer" CDS 7648..7953 /codon_start=1 /gene="ccdB" /product="CcdB, a bacterial toxin that poisons DNA gyrase" /label=ccdB /note="Plasmids containing the ccdB gene cannot be propagated in standard E. coli strains." /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI" primer_bind 7832..7851 /label=ccdB-fwd /note="ccdB gene, forward primer" misc_feature 8388..8505 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 8554..507 /label=hPGK promoter /note="human phosphoglycerate kinase 1 promoter" ORIGIN 1 ttggggttgc gccttttcca aggcagccct gggtttgcgc agggacgcgg ctgctctggg 61 cgtggttccg ggaaacgcag cggcgccgac cctgggtctc gcacattctt cacgtccgtt 121 cgcagcgtca cccggatctt cgccgctacc cttgtgggcc ccccggcgac gcttcctgct 181 ccgcccctaa gtcgggaagg ttccttgcgg ttcgcggcgt gccggacgtg acaaacggaa 241 gccgcacgtc tcactagtac cctcgcagac ggacagcgcc agggagcaat ggcagcgcgc 301 cgaccgcgat gggctgtggc caatagcggc tgctcagcag ggcgcgccga gagcagcggc 361 cgggaagggg cggtgcggga ggcggggtgt ggggcggtag tgtgggccct gttcctgccc 421 gcgcggtgtt ccgcattctg caagcctccg gagcgcacgt cggcagtcgg ctccctcgtt 481 gaccgaatca ccgacctctc tccccagggg gatccaccgg atcaagcttg ccagctgggg 541 cgccctctgg taaggttggg aagccctgca gatctgatcc cggggggcaa tgagatatga 601 aaaagcctga actcaccgcg acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg 661 tctccgacct gatgcagctc tcggagggcg aagaatctcg tgctttcagc ttcgatgtag 721 gagggcgtgg atatgtcctg cgggtaaata gctgcgccga tggtttctac aaagatcgtt 781 atgtttatcg gcactttgca tcggccgcgc tcccgattcc ggaagtgctt gacattgggg 841 aatttagcga gagcctgacc tattgcatct cccgccgtgc acagggtgtc acgttgcaag 901 atctgcctga aaccgaactg cccgctgttc tgcagccggt cgcggaggcc atggatgcga 961 tcgctgcggc cgatcttagc cagacgagcg ggttcggccc attcggaccg caaggaatcg 1021 gtcaatacac tacatggcgt gatttcatat gcgcgattgc tgatccccat gtgtatcact 1081 ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc gcaggctctc gatgagctga 1141 tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca 1201 acaatgtcct gacggacaat ggccgcataa cagcggtcat tgactggagc gaggcgatgt 1261 tcggggattc ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta 1321 tggagcagca gacgcgctac ttcgagcgga ggcatccgga gcttgcagga tcgccgcggc 1381 tccgggcgta tatgctccgc attggtcttg accaactcta tcagagcttg gttgacggca 1441 atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga tccggagccg 1501 ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc gatggctgtg 1561 tagaagtact cgccgatagt ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat 1621 agagtagatg ccgaccggga tctatcgata ccgtcgacct cgaggggggg cccggtacct 1681 ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg 1741 ggactggaag ggctaattca ctcccaacga agaaaagatc tgctttttgc ttgtactggg 1801 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg 1861 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt 1921 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt 1981 agtagttcat gtcatcttat tattcagtat ttataacttg caaagaaatg aatatcagag 2041 agtgagagga acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca 2101 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc 2161 aatgtatctt atcatgtctg gctctagcta tcccgcccct aactccgccc atcccgcccc 2221 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg 2281 cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg 2341 gaggcctagg gacgtaccca attcgcccta tagtgagtcg tattacgcgc gctcactggc 2401 cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc 2461 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc 2521 ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc 2581 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc 2641 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct 2701 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa 2761 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc 2821 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact 2881 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg 2941 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct 3001 tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc 3061 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa 3121 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt 3181 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct 3241 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc 3301 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta 3361 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac 3421 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc 3481 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac 3541 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg 3601 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac 3661 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc 3721 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt 3781 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga 3841 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc 3901 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag 3961 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca 4021 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc 4081 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca 4141 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc 4201 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta 4261 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt 4321 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc 4381 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg 4441 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg 4501 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag 4561 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc 4621 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat 4681 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg 4741 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc 4801 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt 4861 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca 4921 gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg 4981 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac 5041 gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg 5101 gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac 5161 catgattacg ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctgcaa 5221 gcttaatgta gtcttatgca atactcttgt agtcttgcaa catggtaacg atgagttagc 5281 aacatgcctt acaaggagag aaaaagcacc gtgcatgccg attggtggaa gtaaggtggt 5341 acgatcgtgc cttattagga aggcaacaga cgggtctgac atggattgga cgaaccactg 5401 aattgccgca ttgcagagat attgtattta agtgcctagc tcgatacata aacgggtctc 5461 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta 5521 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact 5581 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg 5641 cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg 5701 gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat 5761 tttgactagc ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg 5821 gagaattaga tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga aaaaatataa 5881 attaaaacat atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct 5941 gttagaaaca tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac 6001 aggatcagaa gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca 6061 aaggatagag ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa 6121 aagtaagacc accgcacagc aagcggccgc tgatcttcag acctggagga ggagatatga 6181 gggacaattg gagaagtgaa ttatataaat ataaagtagt aaaaattgaa ccattaggag 6241 tagcacccac caaggcaaag agaagagtgg tgcagagaga aaaaagagca gtgggaatag 6301 gagctttgtt ccttgggttc ttgggagcag caggaagcac tatgggcgca gcgtcaatga 6361 cgctgacggt acaggccaga caattattgt ctggtatagt gcagcagcag aacaatttgc 6421 tgagggctat tgaggcgcaa cagcatctgt tgcaactcac agtctggggc atcaagcagc 6481 tccaggcaag aatcctggct gtggaaagat acctaaagga tcaacagctc ctggggattt 6541 ggggttgctc tggaaaactc atttgcacca ctgctgtgcc ttggaatgct agttggagta 6601 ataaatctct ggaacagatt tggaatcaca cgacctggat ggagtgggac agagaaatta 6661 acaattacac aagcttaata cactccttaa ttgaagaatc gcaaaaccag caagaaaaga 6721 atgaacaaga attattggaa ttagataaat gggcaagttt gtggaattgg tttaacataa 6781 caaattggct gtggtatata aaattattca taatgatagt aggaggcttg gtaggtttaa 6841 gaatagtttt tgctgtactt tctatagtga atagagttag gcagggatat tcaccattat 6901 cgtttcagac ccacctccca accccgaggg gacccgacag gcccgaagga atagaagaag 6961 aaggtggaga gagagacaga gacagatcca ttcgattagt gaacggatct cgacggtatc 7021 gatcacgaga ctagcctcga gcggccgccc ccttcaccga gggcctattt cccatgattc 7081 cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg 7141 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt 7201 ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt 7261 atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccggt gtcttcgagg 7321 cttactaaaa gccagataac agtatgcgta tttgcgcgct gatttttgcg gtataagaat 7381 atatactgat atgtataccc gaagtatgtc aaaaagaggt gtgctatgaa gcagcgtatt 7441 acagtgacag ttgacagcga cagctatcag ttgctcaagg catatatgat gtcaatatct 7501 ccggtctggt aagcacaacc atgcagaatg aagcccgtcg tctgcgtgcc gaacgctgga 7561 aagcggaaaa tcaggaaggg atggctgagg tcgcccggtt tattgaaatg aacggctctt 7621 ttgctgacga gaacagggac tggtgaaatg cagtttaagg tttacaccta taaaagagag 7681 agccgttatc gtctgtttgt ggatgtacag agtgatatta ttgacacgcc cgggcgacgg 7741 atggtgatcc ccctggccag tgcacgtctg ctgtcagata aagtctcccg tgaactttac 7801 ccggtggtgc atatcgggga tgaaagctgg cgcatgatga ccaccgatat ggccagtgtg 7861 ccggtctccg ttatcgggga agaagtggct gatctcagcc accgcgaaaa tgacatcaaa 7921 aacgccatta acctgatgtt ctggggaata taaaccctgc aggacgaaga cccgtttgag 7981 agctactgcc atgaggaatg accaccaggc attccgatcc gacgatggac catcaggcca 8041 tcgagctgca gaagtgacga ccacgcactt cggagtgaca gaggaggatc acccctctgg 8101 ccaccagagt agagcatcag cctactccgg acaactacgg aggaccaccc cgtaggagta 8161 gagcgaggag caccagccct cgcgtgtgac gatgacgatc acgcatcgtc acacaaacta 8221 ctcaaatgtc cgaaaggtgg caaacactcc aaagcagcta aacggatcaa acatggcagt 8281 agcaagttca aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct 8341 ttttttgaat tctcgacctc gagacaaatg gcagtattca tccacaattt taaaagaaaa 8401 ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc aacagacata 8461 caaactaaag aattacaaaa acaaattaca aaaattcaaa attttcgggt ttattacagg 8521 gacagcagag atccactttg gccgcggctc gaggggg //