LOCUS Exported 8647 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses two gRNAs targeting MALAT1 promoter. ACCESSION . VERSION . KEYWORDS pDECKO_Malat1_B SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8647) AUTHORS Aparicio-Prat E, Arnan C, Sala I, Bosch N, Guigo R, Johnson R TITLE DECKO: Single-oligo, dual-CRISPR deletion of genomic elements including long non-coding RNAs. JOURNAL BMC Genomics. 2015 Oct 23;16(1):846. doi: 10.1186/s12864-015-2086-z. PUBMED 26493208 REFERENCE 2 (bases 1 to 8647) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8647 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 87..1264 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 318..1255 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 1212..1232 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" CDS 1277..1876 /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(1277..1296) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 1773..1793 /label=Puro-F /note="Puromycin resistance gene, forward primer" misc_feature 1892..2480 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(1945..1965) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(2363..2374) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 2552..2785 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 2863..2984 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2900..2919) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 2954..2973 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 3024..3159 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3086..3105 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(3179..3198) /label=T7 /note="T7 promoter, forward primer" promoter complement(3180..3198) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(3208..3225) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(3208..3224) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3217..3239) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 3366..3821 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3453..3472) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3663..3684 /label=F1ori-F /note="F1 origin, forward primer" promoter 3847..3951 /gene="bla" /label=AmpR promoter CDS 3952..4812 /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(4170..4189) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 4983..5571 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5472..5491 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 5725..5742 /label=L4440 /note="L4440 vector, forward primer" protein_bind 5859..5880 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 5895..5925 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5933..5949 /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 5938..5960 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 5957..5973 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 5957..5973 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 5992..6012 /label=T3 /note="T3 promoter, forward primer" promoter 5994..6012 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 6040..6266 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 6267..6447 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 6494..6619 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 7112..7345 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 7530..7574 /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 7752..7992 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 7752..7772 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 7923..7942 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 8022..8097 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter 8104..8318 /label=H1 promoter /note="human H1 RNA promoter" primer_bind 8239..8258 /label=H1 /note="Human H1 promoter, forward primer" misc_RNA 8347..8422 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" misc_feature 8478..8595 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" ORIGIN 1 cccgggtgca aagatggata aagttttaaa cagagaggaa tctttgcagc taatggacct 61 tctaggtctt gaaaggagtg ggaattggct ccggtgcccg tcagtgggca gagcgcacat 121 cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgatccggt gcctagagaa 181 ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg 241 gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt 301 ttgccgccag aacacaggta agtgccgtgt gtggttcccg cgggcctggc ctctttacgg 361 gttatggccc ttgcgtgcct tgaattactt ccactggctg cagtacgtga ttcttgatcc 421 cgagcttcgg gttggaagtg ggtgggagag ttcgaggcct tgcgcttaag gagccccttc 481 gcctcgtgct tgagttgagg cctggcctgg gcgctggggc cgccgcgtgc gaatctggtg 541 gcaccttcgc gcctgtctcg ctgctttcga taagtctcta gccatttaaa atttttgatg 601 acctgctgcg acgctttttt tctggcaaga tagtcttgta aatgcgggcc aagatctgca 661 cactggtatt tcggtttttg gggccgcggg cggcgacggg gcccgtgcgt cccagcgcac 721 atgttcggcg aggcggggcc tgcgagcgcg gccaccgaga atcggacggg ggtagtctca 781 agctggccgg cctgctctgg tgcctggcct cgcgccgccg tgtatcgccc cgccctgggc 841 ggcaaggctg gcccggtcgg caccagttgc gtgagcggaa agatggccgc ttcccggccc 901 tgctgcaggg agctcaaaat ggaggacgcg gcgctcggga gagcgggcgg gtgagtcacc 961 cacacaaagg aaaagggcct ttccgtcctc agccgtcgct tcatgtgact ccacggagta 1021 ccgggcgccg tccaggcacc tcgattagtt ctcgagcttt tggagtacgt cgtctttagg 1081 ttggggggag gggttttatg cgatggagtt tccccacact gagtgggtgg agactgaagt 1141 taggccagct tggcacttga tgtaattctc cttggaattt gccctttttg agtttggatc 1201 ttggttcatt ctcaagcctc agacagtggt tcaaagtttt tttcttccat ttcaggtgtc 1261 gtgacgtacg gccaccatga ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga 1321 cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca 1381 caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac 1441 gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg ccgtggcggt 1501 ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat 1561 ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc tcctggcgcc 1621 gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggagtctcgc ccgaccacca 1681 gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg 1741 ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc cccttctacg agcggctcgg 1801 cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg 1861 caagcccggt gcctgaacgc gttaagtcga caatcaacct ctggattaca aaatttgtga 1921 aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt 1981 aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa 2041 atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt 2101 gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct 2161 cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg 2221 ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc 2281 ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg 2341 gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct 2401 gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc 2461 cctttgggcc gcctccccgc gtcgacttta agaccaatga cttacaaggc agctgtagat 2521 cttagccact ttttaaaaga aaagggggga ctggaagggc taattcactc ccaacgaaga 2581 caagatctgc tttttgcttg tactgggtct ctctggttag accagatctg agcctgggag 2641 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt 2701 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt 2761 tagtcagtgt ggaaaatctc tagcagtacg tatagtagtt catgtcatct tattattcag 2821 tatttataac ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt tattgcagct 2881 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca 2941 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggctctag 3001 ctatcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc 3061 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg 3121 agctattcca gaagtagtga ggaggctttt ttggaggcct agggacgtac ccaattcgcc 3181 ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga 3241 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg 3301 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga 3361 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt 3421 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct 3481 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg 3541 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag 3601 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa 3661 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga 3721 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa 3781 atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga 3841 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc 3901 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt 3961 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct 4021 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt 4081 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt 4141 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac 4201 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac 4261 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct 4321 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg 4381 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg 4441 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca 4501 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa 4561 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt 4621 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc 4681 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg 4741 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt 4801 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt 4861 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc 4921 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct 4981 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta 5041 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc 5101 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac 5161 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct 5221 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat 5281 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg 5341 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa 5401 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg 5461 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga 5521 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc 5581 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct 5641 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct 5701 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca 5761 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg 5821 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat 5881 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc 5941 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac 6001 cctcactaaa gggaacaaaa gctggagctg caagcttaat gtagtcttat gcaatactct 6061 tgtagtcttg caacatggta acgatgagtt agcaacatgc cttacaagga gagaaaaagc 6121 accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac 6181 agacgggtct gacatggatt ggacgaacca ctgaattgcc gcattgcaga gatattgtat 6241 ttaagtgcct agctcgatac ataaacgggt ctctctggtt agaccagatc tgagcctggg 6301 agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc 6361 ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct 6421 tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg 6481 gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg 6541 cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag 6601 agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat 6661 tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag 6721 ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca 6781 aatactggga cagctacaac catcccttca gacaggatca gaagaactta gatcattata 6841 taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga 6901 agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc 6961 cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata 7021 aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag 7081 tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag 7141 cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat 7201 tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc 7261 tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa 7321 gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca 7381 ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc 7441 acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct 7501 taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata 7561 aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat 7621 tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag 7681 tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc ccaaccccga 7741 ggggacccag agagggccta tttcccatga ttccttcata tttgcatata cgatacaagg 7801 ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta gtacaaaata 7861 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa 7921 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatacatct 7981 tgtggaaagg acgaaacacc ggcaacttcc attttcagtc tgttttagag ctagaaatag 8041 caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt 8101 tttgaacgct gacgtcatca acccgctcca aggaatcgcg ggcccagtgt cactaggcgg 8161 gaacacccag cgcgcgtgcg ccctggcagg aagatggctg tgagggacag gggagtggcg 8221 ccctgcaata tttgcatgtc gctatgtgtt ctgggaaatc accataaacg tgaaatgtct 8281 ttggatttgg gagtcttata agttctgtat gagaccactc tttcccggaa gcctcagctc 8341 gcctgagttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga 8401 aaaagtggca ccgagtcggt gcttttttaa gcttggcgta actagatctt gagacaaatg 8461 gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa 8521 agaatagtag acataatagc aacagacata caaactaaag aattacaaaa acaaattaca 8581 aaaattcaaa attttcgggt ttattacagg gacagcagag atccactttg gcgccggctc 8641 gaggggg //