LOCUS Exported 8327 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION sgRNA1-2XMS2. ACCESSION . VERSION . KEYWORDS pLH-sgRNA1-2XMS2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8327) AUTHORS Ma H, Tu LC, Naseri A, Huisman M, Zhang S, Grunwald D, Pederson T TITLE Multiplexed labeling of genomic loci with dCas9 and engineered sgRNAs using CRISPRainbow. JOURNAL Nat Biotechnol. 2016 Apr 18. doi: 10.1038/nbt.3526. PUBMED 27088723 REFERENCE 2 (bases 1 to 8327) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8327 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(167..187) /label=hU6-F /note="Human U6 promoter, forward primer" CDS complement(485..529) /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 714..947 /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 1440..1565 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" LTR 1612..1792 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" promoter complement(1793..2019) /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" primer_bind complement(2047..2067) /label=T3 /note="T3 promoter, forward primer" promoter complement(2047..2065) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(2086..2102) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2086..2102) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(2099..2121) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 2110..2126 /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(2134..2164) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2179..2200 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(2317..2334) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2488..3076) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2568..2587) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(3247..4107) /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 3870..3889 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(4108..4212) /gene="bla" /label=AmpR promoter rep_origin complement(4238..4693) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4375..4396) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 4587..4606 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4820..4842 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 4834..4851 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 4835..4851 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 4861..4880 /label=T7 /note="T7 promoter, forward primer" promoter 4861..4879 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" rep_origin 4900..5035 /label=SV40 ori /note="SV40 origin of replication" primer_bind complement(4954..4973) /label=SV40pro-F /note="SV40 promoter/origin, forward primer" polyA_signal 5075..5196 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5086..5105) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 5140..5159 /label=SV40pA-R /note="SV40 polyA, reverse primer" LTR 5268..5501 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" primer_bind 5583..5599 /label=pBluescriptKS /note="For pBluescript vector" primer_bind 5584..5600 /label=KS primer /note="common sequencing primer, one of multiple similar variants" CDS complement(5624..6649) /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" promoter complement(6739..7249) /label=hPGK promoter /note="human phosphoglycerate kinase 1 promoter" primer_bind complement(6802..6820) /label=hPGK-F /note="Human PGK promoter, forward primer" misc_feature 7298..7415 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" misc_RNA complement(7494..7512) /label=MS2 stem loop /note="stem loop that binds the bacteriophage MS2 coat protein" misc_RNA complement(7564..7582) /label=MS2 stem loop /note="stem loop that binds the bacteriophage MS2 coat protein" CDS complement(7620..7925) /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 complement(7722..7741) /label=ccdB-fwd /note="ccdB gene, forward primer" promoter complement(8274..187) /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind complement(8324..16) /label=LKO.1 5' /note="Human U6 promoter, forward primer" ORIGIN 1 taagcatatg atagtccatt ttaaaacata attttaaaac tgcaaactac ccaagaaatt 61 attactttct acgtcacgta ttttgtacta atatctttgt gtttacagtc aaattaattc 121 caattatctc tctaacagcc ttgtatcgta tatgcaaata tgaaggaatc atgggaaata 181 ggccctcggt gaagggggcg gccgctcgag gctagtctcg tgatcgatac cgtcgagatc 241 cgttcactaa tcgaatggat ctgtctctgt ctctctctcc accttcttct tctattcctt 301 cgggcctgtc gggtcccctc ggggttggga ggtgggtctg aaacgataat ggtgaatatc 361 cctgcctaac tctattcact atagaaagta cagcaaaaac tattcttaaa cctaccaagc 421 ctcctactat cattatgaat aattttatat accacagcca atttgttatg ttaaaccaat 481 tccacaaact tgcccattta tctaattcca ataattcttg ttcattcttt tcttgctggt 541 tttgcgattc ttcaattaag gagtgtatta agcttgtgta attgttaatt tctctgtccc 601 actccatcca ggtcgtgtga ttccaaatct gttccagaga tttattactc caactagcat 661 tccaaggcac agcagtggtg caaatgagtt ttccagagca accccaaatc cccaggagct 721 gttgatcctt taggtatctt tccacagcca ggattcttgc ctggagctgc ttgatgcccc 781 agactgtgag ttgcaacaga tgctgttgcg cctcaatagc cctcagcaaa ttgttctgct 841 gctgcactat accagacaat aattgtctgg cctgtaccgt cagcgtcatt gacgctgcgc 901 ccatagtgct tcctgctgct cccaagaacc caaggaacaa agctcctatt cccactgctc 961 ttttttctct ctgcaccact cttctctttg ccttggtggg tgctactcct aatggttcaa 1021 tttttactac tttatattta tataattcac ttctccaatt gtccctcata tctcctcctc 1081 caggtctgaa gatcagcggc cgcttgctgt gcggtggtct tacttttgtt ttgctcttcc 1141 tctatcttgt ctaaagcttc cttggtgtct tttatctcta tcctttgatg cacacaatag 1201 agggttgcta ctgtattata taatgatcta agttcttctg atcctgtctg aagggatggt 1261 tgtagctgtc ccagtatttg tctacagcct tctgatgttt ctaacaggcc aggattaact 1321 gcgaatcgtt ctagctccct gcttgcccat actatatgtt ttaatttata ttttttcttt 1381 ccccctggcc ttaaccgaat tttttcccat cgcgatctaa ttctcccccg cttaatactg 1441 acgctctcgc acccatctct ctccttctag cctccgctag tcaaaatttt tggcgtactc 1501 accagtcgcc gcccctcgcc tcttgccgtg cgcgcttcag caagccgagt cctgcgtcga 1561 gagagctcct ctggtttccc tttcgctttc aagtccctgt tcgggcgcca ctgctagaga 1621 ttttccacac tgactaaaag ggtctgaggg atctctagtt accagagtca cacaacagac 1681 gggcacacac tacttgaagc actcaaggca agctttattg aggcttaagc agtgggttcc 1741 ctagttagcc agagagctcc caggctcaga tctggtctaa ccagagagac ccgtttatgt 1801 atcgagctag gcacttaaat acaatatctc tgcaatgcgg caattcagtg gttcgtccaa 1861 tccatgtcag acccgtctgt tgccttccta ataaggcacg atcgtaccac cttacttcca 1921 ccaatcggca tgcacggtgc tttttctctc cttgtaaggc atgttgctaa ctcatcgtta 1981 ccatgttgca agactacaag agtattgcat aagactacat taagcttgca gctccagctt 2041 ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc 2101 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg 2161 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc 2221 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg 2281 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc 2341 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac 2401 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa 2461 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca 2521 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc 2581 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata 2641 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta 2701 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca 2761 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga 2821 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg 2881 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg 2941 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg 3001 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag 3061 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa 3121 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat 3181 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc 3241 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc 3301 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc 3361 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc 3421 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc 3481 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt 3541 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc 3601 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa 3661 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt 3721 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg 3781 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc 3841 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa 3901 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt 3961 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt 4021 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag 4081 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta 4141 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat 4201 aggggttccg cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt taatattttg 4261 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc 4321 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt 4381 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc 4441 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg 4501 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga 4561 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg 4621 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg 4681 ctacagggcg cgtcccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg 4741 cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt 4801 tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgagcgcgcg 4861 taatacgact cactataggg cgaattgggt acgtccctag gcctccaaaa aagcctcctc 4921 actacttctg gaatagctca gaggccgagg cggcctcggc ctctgcataa ataaaaaaaa 4981 ttagtcagcc atggggcgga gaatgggcgg aactgggcgg agttaggggc gggatgggcg 5041 gagttagggg cgggatagct agagccagac atgataagat acattgatga gtttggacaa 5101 accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct 5161 ttatttgtaa ccattataag ctgcaataaa caagttcctc tcactctctg atattcattt 5221 ctttgcaagt tataaatact gaataataag atgacatgaa ctactactgc tagagatttt 5281 ccacactgac taaaagggtc tgagggatct ctagttacca gagtcacaca acagacgggc 5341 acacactact tgaagcactc aaggcaagct ttattgaggc ttaagcagtg ggttccctag 5401 ttagccagag agctcccagg ctcagatctg gtctaaccag agagacccag tacaagcaaa 5461 aagcagatct tttcttcgtt gggagtgaat tagcccttcc agtcccccct tttcttttaa 5521 aaagtggcta agatctacag ctgccttgta agtcattggt cttaaaggta ccgggccccc 5581 cctcgaggtc gacggtatcg atagatcccg gtcggcatct actctattcc tttgccctcg 5641 gacgagtgct ggggcgtcgg tttccactat cggcgagtac ttctacacag ccatcggtcc 5701 agacggccgc gcttctgcgg gcgatttgtg tacgcccgac agtcccggct ccggatcgga 5761 cgattgcgtc gcatcgaccc tgcgcccaag ctgcatcatc gaaattgccg tcaaccaagc 5821 tctgatagag ttggtcaaga ccaatgcgga gcatatacgc ccggagccgc ggcgatcctg 5881 caagctccgg atgcctccgc tcgaagtagc gcgtctgctg ctccatacaa gccaaccacg 5941 gcctccagaa gaagatgttg gcgacctcgt attgggaatc cccgaacatc gcctcgctcc 6001 agtcaatgac cgctgttatg cggccattgt ccgtcaggac attgttggag ccgaaatccg 6061 cgtgcacgag gtgccggact tcggggcagt cctcggccca aagcatcagc tcatcgagag 6121 cctgcgcgac ggacgcactg acggtgtcgt ccatcacagt ttgccagtga tacacatggg 6181 gatcagcaat cgcgcatatg aaatcacgcc atgtagtgta ttgaccgatt ccttgcggtc 6241 cgaatgggcc gaacccgctc gtctggctaa gatcggccgc agcgatcgca tccatggcct 6301 ccgcgaccgg ctgcagaaca gcgggcagtt cggtttcagg cagatcttgc aacgtgacac 6361 cctgtgcacg gcgggagatg caataggtca ggctctcgct aaattcccca atgtcaagca 6421 cttccggaat cgggagcgcg gccgatgcaa agtgccgata aacataacga tctttgtaga 6481 aaccatcggc gcagctattt acccgcagga catatccacg ccctcctaca tcgaagctga 6541 aagcacgaga ttcttcgccc tccgagagct gcatcaggtc ggagacgctg tcgaactttt 6601 cgatcagaaa cttctcgaca gacgtcgcgg tgagttcagg ctttttcata tctcattgcc 6661 ccccgggatc agatctgcag ggcttcccaa ccttaccaga gggcgcccca gctggcaagc 6721 ttgatccggt ggatccccct ggggagagag gtcggtgatt cggtcaacga gggagccgac 6781 tgccgacgtg cgctccggag gcttgcagaa tgcggaacac cgcgcgggca ggaacagggc 6841 ccacactacc gccccacacc ccgcctcccg caccgcccct tcccggccgc tgctctcggc 6901 gcgccctgct gagcagccgc tattggccac agcccatcgc ggtcggcgcg ctgccattgc 6961 tccctggcgc tgtccgtctg cgagggtact agtgagacgt gcggcttccg tttgtcacgt 7021 ccggcacgcc gcgaaccgca aggaaccttc ccgacttagg ggcggagcag gaagcgtcgc 7081 cggggggccc acaagggtag cggcgaagat ccgggtgacg ctgcgaacgg acgtgaagaa 7141 tgtgcgagac ccagggtcgg cgccgctgcg tttcccggaa ccacgcccag agcagccgcg 7201 tccctgcgca aacccagggc tgccttggaa aaggcgcaac cccaaccccc tcgagccgcg 7261 gccaaagtgg atctctgctg tccctgtaat aaacccgaaa attttgaatt tttgtaattt 7321 gtttttgtaa ttctttagtt tgtatgtctg ttgctattat gtctactatt ctttcccctg 7381 cactgtaccc cccaatcccc ccttttcttt taaaattgtg gatgaatact gccatttgtc 7441 tcgaggtcga gaattcaaaa aaagcaccga ctcggtgcca cttggccctg cagacatggg 7501 tgatcctcat gttggccaag ttgataacgg actagcctta tttgaacttg ctaggccctg 7561 cagacatggg tgatcctcat gttggcctag ctctcaaacg ggtcttcgtc ctgcagggtt 7621 tatattcccc agaacatcag gttaatggcg tttttgatgt cattttcgcg gtggctgaga 7681 tcagccactt cttccccgat aacggagacc ggcacactgg ccatatcggt ggtcatcatg 7741 cgccagcttt catccccgat atgcaccacc gggtaaagtt cacgggagac tttatctgac 7801 agcagacgtg cactggccag ggggatcacc atccgtcgcc cgggcgtgtc aataatatca 7861 ctctgtacat ccacaaacag acgataacgg ctctctcttt tataggtgta aaccttaaac 7921 tgcatttcac cagtccctgt tctcgtcagc aaaagagccg ttcatttcaa taaaccgggc 7981 gacctcagcc atcccttcct gattttccgc tttccagcgt tcggcacgca gacgacgggc 8041 ttcattctgc atggttgtgc ttaccagacc ggagatattg acatcatata tgccttgagc 8101 aactgatagc tgtcgctgtc aactgtcact gtaatacgct gcttcatagc acacctcttt 8161 ttgacatact tcgggtatac atatcagtat atattcttat accgcaaaaa tcagcgcgca 8221 aatacgcata ctgttatctg gcttttagta agcctcgaag acaccggtgt ttcgtccttt 8281 ccacaagata tataaagcca agaaatcgaa atactttcaa gttacgg //