LOCUS Exported 7084 bp ds-DNA circular SYN 06-JUN-2022 DEFINITION For mammalian expression of shRNA: GAGCAGAAGAGGATGAATTTA that targets human PPM1B. ACCESSION . VERSION . KEYWORDS pLKO-puro-hPPM1B SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7084) TITLE Najafov lab plasmids REFERENCE 2 (bases 1 to 7084) AUTHORS . TITLE Direct Submission JOURNAL Exported Jun 6, 2022 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7084 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 42..59 /label=L4440 /note="L4440 vector, forward primer" protein_bind 176..197 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 212..242 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 250..266 /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 255..277 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 274..290 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 274..290 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 309..329 /label=T3 /note="T3 promoter, forward primer" promoter 311..329 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 357..583 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 584..764 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 811..936 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1429..1662 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 1847..1891 /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 2189..2429 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 2189..2209 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 2360..2379 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_feature 2534..2651 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 2700..3210 /label=hPGK promoter /note="human phosphoglycerate kinase 1 promoter" primer_bind 3129..3147 /label=hPGK-F /note="Human PGK promoter, forward primer" CDS 3232..3831 /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(3232..3251) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 3728..3748 /label=Puro-F /note="Puromycin resistance gene, forward primer" LTR 3959..4192 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 4264..4385 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4301..4320) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4355..4374 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 4425..4560 /label=SV40 ori /note="SV40 origin of replication" primer_bind 4487..4506 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(4580..4599) /label=T7 /note="T7 promoter, forward primer" promoter complement(4581..4599) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(4609..4626) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(4609..4625) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4618..4640) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 4767..5222 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4854..4873) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5064..5085 /label=F1ori-F /note="F1 origin, forward primer" promoter 5248..5352 /gene="bla" /label=AmpR promoter CDS 5353..6213 /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(5571..5590) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6384..6972 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6873..6892 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 61 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 121 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 181 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 241 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 301 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctgcaa gcttaatgta 361 gtcttatgca atactcttgt agtcttgcaa catggtaacg atgagttagc aacatgcctt 421 acaaggagag aaaaagcacc gtgcatgccg attggtggaa gtaaggtggt acgatcgtgc 481 cttattagga aggcaacaga cgggtctgac atggattgga cgaaccactg aattgccgca 541 ttgcagagat attgtattta agtgcctagc tcgatacata aacgggtctc tctggttaga 601 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata 661 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta 721 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg 781 gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa 841 gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc 901 ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga 961 tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat 1021 atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca 1081 tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa 1141 gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag 1201 ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc 1261 accgcacagc aagcggccgc tgatcttcag acctggagga ggagatatga gggacaattg 1321 gagaagtgaa ttatataaat ataaagtagt aaaaattgaa ccattaggag tagcacccac 1381 caaggcaaag agaagagtgg tgcagagaga aaaaagagca gtgggaatag gagctttgtt 1441 ccttgggttc ttgggagcag caggaagcac tatgggcgca gcgtcaatga cgctgacggt 1501 acaggccaga caattattgt ctggtatagt gcagcagcag aacaatttgc tgagggctat 1561 tgaggcgcaa cagcatctgt tgcaactcac agtctggggc atcaagcagc tccaggcaag 1621 aatcctggct gtggaaagat acctaaagga tcaacagctc ctggggattt ggggttgctc 1681 tggaaaactc atttgcacca ctgctgtgcc ttggaatgct agttggagta ataaatctct 1741 ggaacagatt tggaatcaca cgacctggat ggagtgggac agagaaatta acaattacac 1801 aagcttaata cactccttaa ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga 1861 attattggaa ttagataaat gggcaagttt gtggaattgg tttaacataa caaattggct 1921 gtggtatata aaattattca taatgatagt aggaggcttg gtaggtttaa gaatagtttt 1981 tgctgtactt tctatagtga atagagttag gcagggatat tcaccattat cgtttcagac 2041 ccacctccca accccgaggg gacccgacag gcccgaagga atagaagaag aaggtggaga 2101 gagagacaga gacagatcca ttcgattagt gaacggatct cgacggtatc gatcacgaga 2161 ctagcctcga gcggccgccc ccttcaccga gggcctattt cccatgattc cttcatattt 2221 gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg taaacacaaa 2281 gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt 2341 aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt 2401 cttggcttta tatatcttgt ggaaaggacg aaacaccggg agcagaagag gatgaattta 2461 ctcgagtaaa ttcatcctct tctgctcttt ttgaattctc gacctcgaga caaatggcag 2521 tattcatcca caattttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa 2581 tagtagacat aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa 2641 ttcaaaattt tcgggtttat tacagggaca gcagagatcc actttggccg cggctcgagg 2701 gggttggggt tgcgcctttt ccaaggcagc cctgggtttg cgcagggacg cggctgctct 2761 gggcgtggtt ccgggaaacg cagcggcgcc gaccctgggt ctcgcacatt cttcacgtcc 2821 gttcgcagcg tcacccggat cttcgccgct acccttgtgg gccccccggc gacgcttcct 2881 gctccgcccc taagtcggga aggttccttg cggttcgcgg cgtgccggac gtgacaaacg 2941 gaagccgcac gtctcactag taccctcgca gacggacagc gccagggagc aatggcagcg 3001 cgccgaccgc gatgggctgt ggccaatagc ggctgctcag cagggcgcgc cgagagcagc 3061 ggccgggaag gggcggtgcg ggaggcgggg tgtggggcgg tagtgtgggc cctgttcctg 3121 cccgcgcggt gttccgcatt ctgcaagcct ccggagcgca cgtcggcagt cggctccctc 3181 gttgaccgaa tcaccgacct ctctccccag ggggatccac cggagcttac catgaccgag 3241 tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc 3301 gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc 3361 gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag 3421 gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa 3481 gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg 3541 gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg 3601 ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc 3661 gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc 3721 gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag 3781 gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg acgcccgccc 3841 cacgacccgc agcgcccgac cgaaaggagc gcacgacccc atgcatcggt acctttaaga 3901 ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg 3961 gaagggctaa ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc 4021 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag 4081 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct 4141 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtagtagt 4201 tcatgtcatc ttattattca gtatttataa cttgcaaaga aatgaatatc agagagtgag 4261 aggaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc 4321 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta 4381 tcttatcatg tctggctcta gctatcccgc ccctaactcc gcccatcccg cccctaactc 4441 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg 4501 ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc 4561 tagggacgta cccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt 4621 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca 4681 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca 4741 gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg 4801 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt 4861 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg 4921 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga 4981 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac 5041 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc 5101 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa 5161 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat 5221 ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata 5281 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga 5341 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca 5401 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat 5461 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag 5521 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc 5581 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct 5641 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca 5701 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt 5761 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat 5821 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt 5881 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta 5941 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga 6001 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt 6061 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc 6121 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct 6181 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata 6241 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt 6301 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc 6361 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg 6421 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact 6481 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg 6541 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg 6601 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac 6661 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca 6721 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga 6781 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc 6841 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct 6901 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg 6961 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct 7021 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc 7081 tttg //