LOCUS Exported 6960 bp ds-DNA circular SYN 18-FEB-2022 DEFINITION Expresses puromycin resistance gene and scrambled gRNA for stable integration in mammalian cells; useful as control and template for new gRNAs by mutagenesis.. ACCESSION . VERSION . KEYWORDS pLenti-gRNA-puro SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6960) AUTHORS Sapozhnikov DM, Szyf M TITLE Unraveling the functional role of DNA demethylation at specific promoters by targeted steric blockage of DNA methyltransferase with CRISPR/dCas9. JOURNAL Nat Commun. 2021 Sep 29;12(1):5711. doi: 10.1038/s41467-021-25991-9. PUBMED 34588447 REFERENCE 2 (bases 1 to 6960) AUTHORS . TITLE Direct Submission JOURNAL Exported Feb 18, 2022 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6960 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 1..227 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 228..408 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 455..580 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1073..1306 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 1491..1535 /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" protein_bind 1827..1851 /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" misc_RNA complement(1863..1938) /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter complement(1967..2207) /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind complement(2017..2036) /label=LKO.1 5' /note="Human U6 promoter, forward primer" primer_bind complement(2187..2207) /label=hU6-F /note="Human U6 promoter, forward primer" CDS 2344..2385 /codon_start=1 /product="epitope tag from simian virus 5" /label=V5 tag /translation="GKPIPNPLLGLDST" CDS 2395..2412 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(2435..2452) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" primer_bind complement(2464..2484) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 2469..2798 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 2649..2784 /label=SV40 ori /note="SV40 origin of replication" CDS 2808..3407 /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(2808..2827) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 3304..3324 /label=Puro-F /note="Puromycin resistance gene, forward primer" LTR 3479..3712 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 3784..3905 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3821..3840) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3875..3894 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 3945..4080 /label=SV40 ori /note="SV40 origin of replication" primer_bind complement(4100..4119) /label=T7 /note="T7 promoter, forward primer" promoter complement(4101..4119) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(4129..4146) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(4129..4145) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4138..4160) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 4287..4742 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4374..4393) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4584..4605 /label=F1ori-F /note="F1 origin, forward primer" promoter 4768..4872 /gene="bla" /label=AmpR promoter CDS 4873..5733 /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(5091..5110) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5904..6492 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6393..6412 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 6646..6663 /label=L4440 /note="L4440 vector, forward primer" protein_bind 6780..6801 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 6816..6846 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6854..6870 /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 6859..6881 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 6878..6894 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 6878..6894 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 6913..6933 /label=T3 /note="T3 promoter, forward primer" promoter 6915..6933 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" ORIGIN 1 tgtagtctta tgcaatactc ttgtagtctt gcaacatggt aacgatgagt tagcaacatg 61 ccttacaagg agagaaaaag caccgtgcat gccgattggt ggaagtaagg tggtacgatc 121 gtgccttatt aggaaggcaa cagacgggtc tgacatggat tggacgaacc actgaattgc 181 cgcattgcag agatattgta tttaagtgcc tagctcgata cataaacggg tctctctggt 241 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc 301 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta 361 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa 421 cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga ctcggcttgc 481 tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa aaattttgac 541 tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc gggggagaat 601 tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa 661 acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga 721 aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc 781 agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat 841 agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa 901 gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat atgagggaca 961 attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac 1021 ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt 1081 tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga 1141 cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg 1201 ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg 1261 caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt 1321 gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat 1381 ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt 1441 acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac 1501 aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt 1561 ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag 1621 tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc 1681 agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg 1741 gagagagaga cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcggatcc 1801 agtgtggtgg aattcgccct ttaatgccaa ctttgtacaa gaaagctggg tctagaaaaa 1861 aagcaccgac tcggtgccac tttttcaagt tgataacgga ctagccttat tttaacttgc 1921 tatttctagc tctaaaactg agttagctct ggtagtgcgg tgtttcgtcc tttccacaag 1981 atatataaag ccaagaaatc gaaatacttt caagttacgg taagcatatg atagtccatt 2041 ttaaaacata attttaaaac tgcaaactac ccaagaaatt attactttct acgtcacgta 2101 ttttgtacta atatctttgt gtttacagtc aaattaattc taattatctc tctaacagcc 2161 ttgtatcgta tatgcaaata tgaaggaatc atgggaaata ggccctcttc ctgcccgacc 2221 ttggtaccgg atccagtcga ctgaattggt tcctttaaag cctgcttttt tgtacaaagg 2281 gcgaattctg cagatatcca gcacagtggc ggccgctcga gtctagaggg cccgcggttc 2341 gaaggtaagc ctatccctaa ccctctcctc ggtctcgatt ctacgcgtac cggtcatcat 2401 caccatcacc attgagttta aacctgctga tcagcctcga ctgtgccttc tagtcgaccc 2461 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta 2521 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag 2581 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa 2641 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac 2701 taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt 2761 agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gcttaccatg accgagtaca 2821 agcccacggt gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc accctcgccg 2881 ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga tccggaccgc cacatcgagc 2941 gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt 3001 gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc gtcgaagcgg 3061 gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc cggctggccg 3121 cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc 3181 tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc 3241 tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag acctccgcgc 3301 cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc 3361 ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctgaggt acctttaaga 3421 ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg 3481 gaagggctaa ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc 3541 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag 3601 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct 3661 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtagtagt 3721 tcatgtcatc ttattattca gtatttataa cttgcaaaga aatgaatatc agagagtgag 3781 aggaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc 3841 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta 3901 tcttatcatg tctggctcta gctatcccgc ccctaactcc gcccatcccg cccctaactc 3961 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg 4021 ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc 4081 tagggacgta cccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt 4141 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca 4201 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca 4261 gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg 4321 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt 4381 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg 4441 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga 4501 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac 4561 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc 4621 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa 4681 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat 4741 ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata 4801 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga 4861 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca 4921 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat 4981 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag 5041 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc 5101 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct 5161 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca 5221 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt 5281 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat 5341 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt 5401 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta 5461 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga 5521 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt 5581 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc 5641 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct 5701 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata 5761 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt 5821 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc 5881 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg 5941 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact 6001 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg 6061 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg 6121 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac 6181 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca 6241 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga 6301 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc 6361 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct 6421 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg 6481 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct 6541 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc 6601 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc 6661 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat 6721 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt 6781 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt 6841 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat 6901 tacgccaagc gcgcaattaa ccctcactaa agggaacaaa agctggagct gcaagcttaa //