LOCUS Exported 8276 bp ds-DNA circular SYN 31-OCT-2022 DEFINITION Expresses dox-controlled miR-E shRNAs (UT4GEPIR). Multi-miR shRNAs targeting A-RAF, B-RAF, and C-RAF (shABC2 = shARAF.169-shBRAF.1296-shCRAF.387). The shABC2 set is better than shABC1.. ACCESSION . VERSION . KEYWORDS pCF806-shABC2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8276) AUTHORS Amen AM, Loughran RM, Huang CH, Lew RJ, Ravi A, Guan Y, Schatoff EM, Dow LE, Emerling BM, Fellmann C TITLE Endogenous spacing enables co-processing of microRNAs and efficient combinatorial RNAi. JOURNAL Cell Rep Methods. 2022 Jun 21;2(7):100239. doi: 10.1016/j.crmeth.2022.100239. eCollection 2022 Jul 18. PUBMED 35880017 REFERENCE 2 (bases 1 to 8276) AUTHORS . TITLE Direct Submission JOURNAL Exported Oct 31, 2022 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8276 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 339..362 /label=MSCV-rev /note="Murine stem cell virus, reverse primer" CDS complement(431..457) /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS complement(1547..1600) /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 complement(2084..2107) /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" regulatory 2107..2116 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" promoter complement(2123..2472) /label=TRE3GV promoter /note="3rd-generation Tet-responsive promoter that can be activated by binding of Tet-On(R) 3G, optimized for retroviral and lentiviral vectors" primer_bind complement(2160..2184) /label=LNCX /note="Human CMV promoter, forward primer" protein_bind 2230..2248 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 2266..2284 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 2302..2320 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 2338..2356 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 2374..2392 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 2410..2428 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 2446..2464 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" CDS complement(2512..2928) /label=gag (truncated) /note="truncated Moloney murine leukemia virus (MMLV) gag gene lacking the start codon" primer_bind complement(2528..2544) /label=pBABE 5' /note="Psi packaging signal, forward primer for pBABE vectors" primer_bind complement(2560..2582) /label=pLXSN 5' /note="Murine stem cell virus, forward primer. Also called MSCV" misc_feature 3129..3328 /label=MMLV Psi /note="packaging signal of Moloney murine leukemia virus (MMLV)" LTR 3391..3566 /label=5' LTR (truncated) /note="truncated long terminal repeat from Moloney murine sarcoma virus" promoter complement(3567..3770) /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind complement(3596..3616) /label=CMV-F /note="Human CMV immediate early promoter, forward primer" enhancer 3771..4150 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" primer_bind complement(4250..4268) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 4336..4440 /gene="bla" /label=AmpR promoter CDS 4441..5301 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(4659..4678) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5472..6060 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5961..5980 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 6214..6231 /label=L4440 /note="L4440 vector, forward primer" primer_bind complement(6312..6331) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" promoter complement(6353..6682) /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 6367..6502 /label=SV40 ori /note="SV40 origin of replication" primer_bind complement(6421..6440) /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind 6667..6687 /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" primer_bind 6823..6845 /label=pGEX 3' /note="pGEX vectors, reverse primer" LTR 6948..7373 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from Moloney murine leukemia virus" CDS complement(7554..8153) /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(7637..7657) /label=Puro-F /note="Puromycin resistance gene, forward primer" primer_bind 8134..8153 /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" promoter complement(8174..397) /label=PGK promoter /note="mouse phosphoglycerate kinase 1 promoter" primer_bind complement(8222..8241) /label=mPGK-F /note="Mouse PGK promoter, forward primer" ORIGIN 1 cccctgagcn nncccctgag cccgcccccg gacccacccc ttcccagcct ctgagcccag 61 aaagcgaagg agcaaagctg ctattggccg ctgccccaaa ggcctacccg cttccattgc 121 tcagcggtgc tgtccatctg cacgagacta gtgagacgtg ctacttccat ttgtcacgtc 181 ctgcacgacg cgagctgcgg ggcggggggg aacttcctga ctaggggagg agtagaaggt 241 ggcgcgaagg ggccaccaaa gaacggagcc ggttggcgcc taccggtgga tgtggaatgt 301 gtgcgaggcc agaggccact tgtgtagcgc caagtgccca gcggggctgc taaagcgcat 361 gctccagact gccttgggaa aagcgcctcc cctacccggt agaattcggg cccgccggcg 421 cggccgctca agcgtaatct ggaacatcgt atgggtaaaa ttcatcaaaa actggaaact 481 cattgtttct ttcatattca atttgctttg tcagaaattc tttacttctt ttgtcaatta 541 agttgctaga gaatagattg aattcaggaa taaaataatt ctcaagtttt tctgtcctga 601 ggcactgaag aaagtatgtc acgcagttat caaagcagag gcccaggtct ttgcggtccc 661 actgactgtc ttgagggttc tgggtacata cgtgaaagaa ggcagttttc acatgataag 721 aagagaattt atccagatgt tttttgtctt taaacctttc tttcagctgt tctaaaaggt 781 atttcattag ttttaaacaa tctttcctgc aacatttctc ttctttgttt tcacagcacg 841 ttttagattt tccatgattg ttcaaaattt ccttttcgat gtgagagaag gatagccgcc 901 atgtttcttc ttggaaacca tttccttcct ttgcatgctt gggtacaagg taaaatggct 961 ttagtcgtag ttgcttccta acttttgctg aaagccagtt ttgaatgcgc aggccttctt 1021 gggtgctagc aggccagcta ctttttgatt ccaaagccag ggttatatcc acagatattt 1081 tttcactaat aagaagtgtt acagcagggc tccctcctct tttcctcttc atgatgacat 1141 ctgtatcttt aatgtcgtta atttcttcct taatgatttt cctaaacttt gacagcatct 1201 tagaagctga taatatttca ccttctaaaa actgactcag aggattttct ttcggatttc 1261 ttttaaattt cacaaagtaa tatgcacgag tgttggaata ttcttctagt tgaattctgg 1321 ggacttccag tttaaacatg acatcaaatt cattaggtgc agaaatcttc acgtgctcat 1381 agtagctccc ggtgttcagc agcccgacgc ctctgaacgc ggagtcgcac ttcagtctga 1441 gcagcaggtg gtccacaacc cctttcacca tccccgccgc cgtggagata tcatcgcggc 1501 tgagcttcaa cttctccaaa accgcccgga gcttcgaggc cccaggaggg ccgggattct 1561 cttccacgtc accgcatgtc agcagactgc ctctgccctc tccgctaccc gccgcatccc 1621 tccgtacgag aatgggggcc gagaccggca ggccggggct gggcacgtcc cagggcccgg 1681 gcggaggtct tggcttcgtg gagcagcgcg cgcccctctg gcggcaagaa ccagccctgg 1741 aaagagccgg ctcccgagcc gcaggaggct ccagcccctc tgccccaggg gcgctggtgg 1801 cgtcagacgg ctgcgtgtcc tgggcgcgct gaggggcctt tttggcgcgg gccccagttg 1861 cgcggacggg cggcctctcc tgggtgtccg gggcgctctt tttctgccgg gatcccgact 1921 tcctggcggg gccgaacttt cccgccttag gcagggcggc ctcgggggca gccggagact 1981 cggtgggatc catcggggcg cccctggcat tccgtgcgga agccttgggg gcagtggctc 2041 cggcctcgga agctctctgc atggcctttc cgtgccaagg ctgcttatcg tcgtcatcct 2101 tgtaatccat ggtggcggat ccaagtataa gacaaaagtg ttgtggaatt gctccaggcg 2161 atctgacggt tcactaaacg agctctgctt ttataggcgc ccaccgtaca cgcctacctc 2221 gacatacgtt ctctatcact gatagggagt aaactggata tacgttctct atcactgata 2281 gggagtaaac tgtagatacg ttctctatca ctgataggga gtaaactggt catacgttct 2341 ctatcactga tagggagtaa actccttata cgttctctat cactgatagg gagtaaagtc 2401 tgcatacgtt ctctatcact gatagggagt aaactcttca tacgttctct atcactgata 2461 gggagtaaac tcgagtgaag acgaaagggc ctcatcgaga tcttcaattc cggcgcctag 2521 agaaggagtg agggctggat aaagggagga tcgaggcggg gtcgaacgag gaggttcaag 2581 ggggagagac ggggcggatg gaggaagagg aggcggaggc ttagggtgta caaagggctt 2641 gacccaggga ggggggtcaa aagccaaggc ttcccaggtc acgatgtagg ggacctggtc 2701 tgggtgtcca tgcgggccag gtgaaaagac cttgatctta acctgggtga tgaggtctcg 2761 gttaaaggtg ccgtctcgcg gccatccgac gttaaaggtt ggccattctg cagagcagaa 2821 ggtaacccaa cgtctcttct tgacatctac cgactggttg tgagcgatcc gctcgacatc 2881 tttccagtga cctaaggtca aacttaaggg agtggtaaca gtctggccct aattttcaga 2941 caaatacaga aacacagtca gacagagaca acacagaacg atgctgcagc gctgcagcag 3001 acaagacgcg cggcttcggt tccaaaccga aagcaaaaat tcagacggag gcgggaactg 3061 ttttaggttc tcgtctccta ccagaaccac atatcctgac ggggtcggat tccacatcga 3121 ctcccttcct caggtcgggc cacaaaaacg gcccccaaag tccctgggac gtctcccagg 3181 gttgcggccg ggtgttcaga actcgtcagt tccaccacgg gtccgccaga tacagagcta 3241 gttagctaac tagtacagac gcaggcgcat aacatcaaac atagacacta gacaatcgga 3301 cagacacaga taagttgctg gccagcttac ctcccggtgg tgggtcggtg gtccctgggc 3361 aggggtctcc aaatcccgga cgagccccca aatgaaagac ccccgtcgtg ggtagtcaat 3421 cactcagagg agaccctccc aaggaacagc gagaccacga ttcggatgca aacagcaaga 3481 ggctttattg ggaatacggg tacccgggcg acgcagtcta tcggaagact ggcgcgccga 3541 gtgaggggtt gtgggctctt ttattgagct ctgcttatat agacctccca ccgtacacgc 3601 ctaccgccca tttgcgtcaa tggggcggag ttgttacgac attttggaaa gtcccgttga 3661 ttttggtgcc aaaacaaact cccattgacg tcaatggggt ggagacttgg aaatccccgt 3721 gagtcaaacc gctatccacg cccattgatg tactgccaaa accgcatcac catggtaata 3781 gcgatgacta atacgtagat gtactgccaa gtaggaaagt cccataaggt catgtactgg 3841 gcataatgcc aggcgggcca tttaccgtca ttgacgtcaa tagggggcgt acttggcata 3901 tgatacactt gatgtactgc caagtgggca gtttaccgta aatactccac ccattgacgt 3961 caatggaaag tccctattgg cgttactatg ggaacatacg tcattattga cgtcaatggg 4021 cgggggtcgt tgggcggtca gccaggcggg ccatttaccg taagttatgt aacgcggaac 4081 tccatatatg ggctatgaac taatgacccc gtaattgatt actattaata actagtcaat 4141 aatcaatgtc aacatggcgg taatgttgga catgagccaa tataaatgta catattatga 4201 tatggataca acgtatgcaa tggccaagct aattcttgaa gacgaaaggg cctcgtgata 4261 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact 4321 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg 4381 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt 4441 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct 4501 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca 4561 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc 4621 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc 4681 cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg 4741 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta 4801 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc 4861 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt 4921 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg 4981 cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct 5041 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc 5101 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct 5161 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac 5221 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc 5281 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat 5341 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg 5401 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc 5461 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa 5521 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag 5581 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta 5641 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta 5701 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag 5761 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg 5821 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg 5881 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag 5941 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc 6001 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa 6061 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg 6121 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct 6181 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa 6241 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatgg 6301 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtaagc tttttgcaaa 6361 agcctaggcc tccaaaaaag cctcctcact acttctggaa tagctcagag gccgaggcgg 6421 cctcggcctc tgcataaata aaaaaaatta gtcagccatg gggcggagaa tgggcggaac 6481 tgggcggagt taggggcggg atgggcggag ttaggggcgg gactatggtt gctgactaat 6541 tgagatgcat gctttgcata cttctgcctg ctggggagcc tggggacttt ccacacctgg 6601 ttgctgacta attgagatgc atgctttgca tacttctgcc tgctggggag cctggggact 6661 ttccacaccc taactgacac acattccaca gccggatcta cactccgcta tcgctacgtg 6721 actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt 6781 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc 6841 agaggttttc accgtcatca ccgaaacgcg cgaggcagcc agcttacctc ccggtggtgg 6901 gtcggtggtc cctgggcagg ggtctcccga tcccggacga gcccccaaat gaaagacccc 6961 cgctgacggg tagtcaatca ctcagaggag accctcccaa ggaacagcga gaccacaagt 7021 cggatgcaac tgcaagaggg tttattggat acacgggtac ccgggcgact cagtcaatcg 7081 gaggactggc gccccgagtg aggggttgtg ggctctttta ttgagctcgg ggagcagaag 7141 cgcgcgaaca gaagcgagaa gcgaactgat tggttagttc aaataaggca cagggtcatt 7201 tcaggtcctt ggggcaccct ggaaacatct gatggttctc tagggtcgac cctgttccat 7261 ctgttcctga ccttgatctg aacttctcta ttctcagtta tgtatttttc catgccttgc 7321 aaaatggcgt tacttaagct agcttgccaa acctacaggt ggggtctttc attcccccct 7381 ttttctggag actaaataaa atcttttatt ttatctatgg ctcgtactct ataggcttca 7441 gctggtgata ttgttgagtc aaaactagag cctggaccac tgatatctcg agacgccgat 7501 gcatggggtc gtgcgctcct ttcggtcggg cgctgcgggt cgtggggcgg gcgtcaggca 7561 ccgggcttgc gggtcatgca ccaggtgcgc ggtccttcgg gcacctcgac gtcggcggtg 7621 acggtgaagc cgagccgctc gtagaagggg aggttgcggg gcgcggaggt ctccaggaag 7681 gcgggcaccc cggcgcgctc ggccgcctcc actccgggga gcacgacggc gctgcccaga 7741 cccttgccct ggtggtcggg cgagacgccg acggtggcca ggaaccacgc gggctccttg 7801 ggccggtgcg gcgccaggag gccttccatc tgttgctgcg cggccagccg ggaaccgctc 7861 aactcggcca tgcgcgggcc gatctcggcg aacaccgccc ccgcttcgac gctctccggc 7921 gtggtccaga ccgccaccgc ggcgccgtcg tccgcgaccc acaccttgcc gatgtcgagc 7981 ccgacgcgcg tgaggaagag ttcttgcagc tcggtgaccc gctcgatgtg gcggtccgga 8041 tcgacggtgt ggcgcgtggc ggggtagtcg gcgaacgcgg cggcgagggt gcgtacggcc 8101 ctggggacgt cgtcgcgggt ggcgaggcgc accgtgggct tgtactcggt catggtaagc 8161 ttgggctgca ggtcgaaagg cccggagatg aggaagagga gaacagcgcg gcagacgtgc 8221 gcttttgaag cgtgcagaat gccgggcctc cggaggacct tcgggcgccc gccccg //