LOCUS Exported 8910 bp ds-DNA circular SYN 12-OCT-2021 DEFINITION Expression of CasMINI sgRNA targeting TRE3G promoter in dCasMINI-mediated GFP activation assays.. ACCESSION . VERSION . KEYWORDS pSLQ9834_sgTet: pHR-hU6-CasMINI sgRNA_#2 (sgTet); EF1a-Puro-T2... SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8910) AUTHORS Xu X, Chemparathy A, Zeng L, Kempton HR, Shang S, Nakamura M, Qi LS TITLE Engineered miniature CRISPR-Cas system for mammalian genome regulation and editing. JOURNAL Mol Cell. 2021 Aug 26. pii: S1097-2765(21)00648-1. doi: 10.1016/j.molcel.2021.08.008. PUBMED 34480847 REFERENCE 2 (bases 1 to 8910) AUTHORS . TITLE Direct Submission JOURNAL Exported Oct 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8910 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(47..66) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 238..617 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 619..817 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 772..792 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" LTR 835..1015 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 1062..1187 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1684..1917 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 2102..2146 /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 2444..2561 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 2620..2860 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 2620..2640 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 2791..2810 /label=LKO.1 5' /note="Human U6 promoter, forward primer" protein_bind 3030..3048 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" promoter 3135..3346 /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" intron 3347..4285 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 4242..4262 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" CDS 4320..4916 /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(4320..4339) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 4816..4836 /label=Puro-F /note="Puromycin resistance gene, forward primer" CDS 4926..4979 /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" CDS 4992..5690 /codon_start=1 /product="monomeric blue fluorescent protein" /label=TagBFP /note="mammalian codon-optimized" /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD LPSKLGHKLN" protein_bind 5710..5743 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 5799..6387 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(5852..5872) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(6270..6281) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(6390..6406) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6391..6407) /label=pBluescriptKS /note="For pBluescript vector" LTR 6916..7096 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" rep_origin complement(7158..7746) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(7238..7257) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(7917..8777) /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 8540..8559 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(8778..8882) /gene="bla" /label=AmpR promoter ORIGIN 1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg 61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt 121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc 181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac 241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat 301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg 361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt 421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag 481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc 541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag 601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt 661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc 721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg 781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct 841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt 901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac 961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc 1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc 1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa 1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg 1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata 1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc 1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga 1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc 1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca 1501 aaagtaagac caccgcacag caagcggccg gccgctgatc ttcagacctg gaggaggaga 1561 tatgagggac aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt 1621 aggagtagca cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg 1681 aataggagct ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc 1741 aatgacgctg acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa 1801 tttgctgagg gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa 1861 gcagctccag gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg 1921 gatttggggt tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg 1981 gagtaataaa tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga 2041 aattaacaat tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga 2101 aaagaatgaa caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa 2161 cataacaaat tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg 2221 tttaagaata gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc 2281 attatcgttt cagacccacc tcccaacccc gaggggaccc gacaggcccg aaggaataga 2341 agaagaaggt ggagagagag acagagacag atccattcga ttagtgaacg gatcggcact 2401 gcgtgcgcca attctgcaga caaatggcag tattcatcca caattttaaa agaaaagggg 2461 ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa 2521 ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca 2581 gcagagatcc agtttggtta gtaccgggcc cgctctagag agggcctatt tcccatgatt 2641 ccttcatatt tgcatatacg atacaaggct gttagagaga taattagaat taatttgact 2701 gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag 2761 tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag 2821 tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccgg gcttcactga 2881 taaagtggag aaccgcttca ccaaaagctg tcccttaggg gattagaact tgagtgaagg 2941 tgggctgctt gcatcagcct aatgtcgaga agtgctttct tcggaaagta accctcgaaa 3001 caaattcatt tgaatgaagg aatgcaacct ccctatcagt gatagagaac gtttttttaa 3061 gcttgggccg ctcgagggat ccattaggcg gccgcgtgga taaccgtatt accgccatgc 3121 atgtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg 3181 ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt 3241 cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt 3301 cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggtaa gtgccgtgtg 3361 tggttcccgc gggcctggcc tctttacggg ttatggccct tgcgtgcctt gaattacttc 3421 cacctggctg cagtacgtga ttcttgatcc cgagcttcgg gttggaagtg ggtgggagag 3481 ttcgaggcct tgcgcttaag gagccccttc gcctcgtgct tgagttgagg cctggcctgg 3541 gcgctggggc cgccgcgtgc gaatctggtg gcaccttcgc gcctgtctcg ctgctttcga 3601 taagtctcta gccatttaaa atttttgatg acctgctgcg acgctttttt tctggcaaga 3661 tagtcttgta aatgcgggcc aagatctgca cactggtatt tcggtttttg gggccgcggg 3721 cggcgacggg gcccgtgcgt cccagcgcac atgttcggcg aggcggggcc tgcgagcgcg 3781 gccaccgaga atcggacggg ggtagtctca agctggccgg cctgctctgg tgcctggcct 3841 cgcgccgccg tgtatcgccc cgccctgggc ggcaaggctg gcccggtcgg caccagttgc 3901 gtgagcggaa agatggccgc ttcccggccc tgctgcaggg agctcaaaat ggaggacgcg 3961 gcgctcggga gagcgggcgg gtgagtcacc cacacaaagg aaaagggcct ttccgtcctc 4021 agccgtcgct tcatgtgact ccacggagta ccgggcgccg tccaggcacc tcgattagtt 4081 ctcgcgcttt tggagtacgt cgtctttagg ttggggggag gggttttatg cgatggagtt 4141 tccccacact gagtgggtgg agactgaagt taggccagct tggcacttga tgtaattctc 4201 cttggaattt gccctttttg agtttggatc ttggttcatt ctcaagcctc agacagtggt 4261 tcaaagtttt tttcttccat ttcaggtgtc gtgagctagc ccaccatgac cgacccacca 4321 tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc cgggccgtac 4381 gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gacccggacc 4441 gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca 4501 tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc acgccggaga 4561 gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt 4621 cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc 4681 ccgcgtggtt cctggccacc gtcggcgttt ctccagacca ccagggcaag ggtctgggca 4741 gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg 4801 agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc gtcaccgccg 4861 acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgccggat 4921 cgggagaggg cagaggaagt ctgctaacat gcggtgacgt cgaggagaat cctggcccac 4981 cggtcgccac cagcgagctg attaaggaga acatgcacat gaagctgtac atggagggca 5041 ccgtggacaa ccatcacttc aagtgcacat ccgagggcga aggcaagccc tacgagggca 5101 cccagaccat gagaatcaag gtggtcgagg gcggccctct ccccttcgcc ttcgacatcc 5161 tggctactag cttcctctac ggcagcaaga ccttcatcaa ccacacccag ggcatccccg 5221 acttcttcaa gcagtccttc cctgagggct tcacatggga gagagtcacc acatacgaag 5281 acgggggcgt gctgaccgct acccaggaca ccagcctcca ggacggctgc ctcatctaca 5341 acgtcaagat cagaggggtg aacttcacat ccaacggccc tgtgatgcag aagaaaacac 5401 tcggctggga ggccttcacc gagactctgt atcctgctga cggcggcctg gaaggcagaa 5461 acgacatggc cctgaagctc gtgggcggga gccatctgat cgcaaacatc aagaccacat 5521 atagatccaa gaaacccgct aagaacctca agatgcctgg cgtctactat gtggactaca 5581 gactggaaag aatcaaggag gccaacaacg agacctacgt cgagcagcac gaggtggcag 5641 tggccagata ctgcgacctc cctagcaaac tggggcacaa gcttaattaa gaattcgtcg 5701 agggacctaa taacttcgta tagcatacat tatacgaagt tatacatgtt taagggttcc 5761 ggttccacta ggtacaattc gatatcaagc ttatcgataa tcaacctctg gattacaaaa 5821 tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg 5881 ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct 5941 tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg 6001 gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct 6061 gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg 6121 ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg 6181 tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc 6241 tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc 6301 gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc 6361 ggatctccct ttgggccgcc tccccgcatc gataccgtcg acctcgatcg agacctagaa 6421 aaacatggag caatcacaag tagcaataca gcagctacca atgctgattg tgcctggcta 6481 gaagcacaag aggaggagga ggtgggtttt ccagtcacac ctcaggtacc tttaagacca 6541 atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa 6601 gggctaattc actcccaacg aagacaagat atccttgatc tgtggatcta ccacacacaa 6661 ggctacttcc ctgattggca gaactacaca ccagggccag ggatcagata tccactgacc 6721 tttggatggt gctacaagct agtaccagtt gagcaagaga aggtagaaga agccaatgaa 6781 ggagagaaca cccgcttgtt acaccctgtg agcctgcatg ggatggatga cccggagaga 6841 gaagtattag agtggaggtt tgacagccgc ctagcatttc atcacatggc ccgagagctg 6901 catccggact gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc 6961 taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg 7021 tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg 7081 tggaaaatct ctagcagcat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 7141 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 7201 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 7261 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 7321 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg 7381 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 7441 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 7501 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 7561 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct 7621 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 7681 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 7741 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 7801 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat 7861 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac 7921 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt 7981 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt 8041 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag 8101 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct 8161 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt 8221 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc 8281 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt 8341 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg 8401 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg 8461 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct 8521 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc 8581 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt 8641 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt 8701 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg 8761 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat 8821 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg 8881 cgcacatttc cccgaaaagt gccacctgac //