LOCUS Exported 7479 bp ds-DNA circular SYN 14-FEB-2022 DEFINITION Retrovirus for delivery of one sgRNA (empty, bbsI). ACCESSION . VERSION . KEYWORDS pMSCV-U6sgRNA(BbsI)-PGKpuro2ABFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7479) AUTHORS Henriksson J, Chen X, Gomes T, Ullah U, Meyer KB, Miragaia R, Duddy G, Pramanik J, Yusa K, Lahesmaa R, Teichmann SA TITLE Genome-wide CRISPR Screens in T Helper Cells Reveal Pervasive Crosstalk between Activation and Differentiation. JOURNAL Cell. 2019 Jan 7. pii: S0092-8674(18)31569-1. doi: 10.1016/j.cell.2018.11.044. PUBMED 30639098 REFERENCE 2 (bases 1 to 7479) AUTHORS . TITLE Direct Submission JOURNAL Exported Feb 14, 2022 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7479 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 75..94 /label=mPGK-F /note="Mouse PGK promoter, forward primer" CDS 182..778 /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(182..201) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 678..698 /label=Puro-F /note="Puromycin resistance gene, forward primer" CDS 797..850 /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 851..1552 /codon_start=1 /product="monomeric blue fluorescent protein" /label=TagBFP /note="mammalian codon-optimized" /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC DLPSKLGHKLN" LTR 1638..2152 /label=3' LTR /note="3' long terminal repeat from murine embryonic stem cell virus" protein_bind 2314..2330 /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)." promoter complement(2338..2368) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2383..2404 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(2521..2538) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2692..3280) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2772..2791) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(3451..4311) /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 4074..4093 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(4312..4416) /gene="bla" /label=AmpR promoter primer_bind 4484..4502 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(4540..4562) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 4662..4681 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 4875..4897 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 5025..5044 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" LTR 5244..5760 /label=5' LTR /note="5' long terminal repeat from murine embryonic stem cell virus" misc_feature 5824..6165 /label=MESV Psi /note="packaging signal of murine embryonic stem cell virus" CDS 6232..6648 /label=gag (truncated) /note="truncated Moloney murine leukemia virus (MMLV) gag gene lacking the start codon" primer_bind 6578..6600 /label=pLXSN 5' /note="Murine stem cell virus, forward primer. Also called MSCV" primer_bind 6616..6632 /label=pBABE 5' /note="Psi packaging signal, forward primer for pBABE vectors" promoter 6759..6999 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 6759..6779 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 6930..6949 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 7024..7099 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter 7122..142 /label=PGK promoter /note="mouse phosphoglycerate kinase 1 promoter" primer_bind complement(7157..7180) /label=MSCV-rev /note="Murine stem cell virus, reverse primer" ORIGIN 1 gggtgggtcc gggggcgggc tcaggggcgg gctcaggggc ggggcgggcg cccgaaggtc 61 ctccggaggc ccggcattct gcacgcttca aaagcgcacg tctgccgcgc tgttctcctc 121 ttcctcatct ccgggccttt cgacctgcat ccatctagat ctcgagcagc tgaagcttac 181 catgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt 241 acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga 301 ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga 361 catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga 421 gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg 481 ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga 541 gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg 601 cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct 661 ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc 721 cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgccgg 781 cggcgggtcc ggaggagagg gcagaggaag tctcctaaca tgcggtgacg tggaggagaa 841 tcctggccca atgagcgagc tgattaagga gaacatgcac atgaagctgt acatggaggg 901 caccgtggac aaccatcact tcaagtgcac atccgagggc gaaggcaagc cctacgaggg 961 cacccagacc atgagaatca aggtggtcga gggcggccct ctccccttcg ccttcgacat 1021 cctggctact agcttcctct acggcagcaa gaccttcatc aaccacaccc agggcatccc 1081 cgacttcttc aagcagtcct tccctgaggg cttcacatgg gagagagtca ccacatacga 1141 ggacgggggc gtgctgaccg ctacccagga caccagcctc caggacggct gcctcatcta 1201 caacgtcaag atcagagggg tgaacttcac atccaacggc cctgtgatgc agaagaaaac 1261 actcggctgg gaggccttca ccgagacgct gtaccccgct gacggcggcc tggaaggcag 1321 aaacgacatg gccctgaagc tcgtgggcgg gagccatctg atcgcaaaca tcaagaccac 1381 atatagatcc aagaaacccg ctaagaacct caagatgcct ggcgtctact atgtggacta 1441 cagactggaa agaatcaagg aggccaacaa cgagacctac gtcgagcagc acgaggtggc 1501 agtggccaga tactgcgacc tccctagcaa actggggcac aagcttaatt gagcggccgc 1561 taggtacctt taagaccaat gacttacaag gatcgataaa ataaaagatt ttatttagtc 1621 tccagaaaaa ggggggaatg aaagacccca cctgtaggtt tggcaagcta gcttaagtaa 1681 cgccattttg caaggcatgg aaaatacata actgagaata gagaagttca gatcaaggtt 1741 aggaacagag agacagcaga atatgggcca aacaggatat ctgtggtaag cagttcctgc 1801 cccggctcag ggccaagaac agatggtccc cagatgcggt cccgccctca gcagtttcta 1861 gagaaccatc agatgtttcc agggtgcccc aaggacctga aatgaccctg tgccttattt 1921 gaactaacca atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa 1981 taaaagagcc cacaacccct cactcggcgc gccagtcctc cgatagactg cgtcgcccgg 2041 gtacccgtgt atccaataaa ccctcttgca gttgcatccg acttgtggtc tcgctgttcc 2101 ttgggagggt ctcctctgag tgattgacta cccgtcagcg ggggtctttc atgggtaaca 2161 gtttcttgaa gttggagaac aacattctga gggtaggagt cgaatattaa gtaatcctga 2221 ctcaattagc cactgttttg aatccacata ctccaatact cctgaaatag ttcattatgg 2281 acagcgcaga aagagctggg gagaattgtg aaattgttat ccgctcacaa ttccacacaa 2341 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac 2401 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca 2461 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc 2521 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc 2581 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc 2641 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag 2701 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc 2761 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt 2821 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct 2881 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg 2941 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct 3001 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat 3061 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg 3121 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa 3181 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt 3241 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 3301 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt 3361 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta 3421 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat 3481 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac 3541 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg 3601 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag 3661 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt 3721 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt 3781 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt 3841 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt 3901 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct 3961 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt 4021 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac 4081 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa 4141 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa 4201 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca 4261 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct 4321 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga 4381 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc 4441 tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag 4501 gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc 4561 ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc 4621 gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt 4681 actgagagtg caccatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg 4741 catcaggcgc cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc 4801 ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt 4861 aacgccaggg ttttcccagt cacgacgttg taaaacgacg gcgcaaggaa tggtgcatgc 4921 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa 4981 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata 5041 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag 5101 aggcgattag tccaatttgt taaagacagg atatcagtgg tccaggctct agttttgact 5161 caacaatatc accagctgaa gcctatagag tacgagccat agataaaata aaagatttta 5221 tttagtctcc agaaaaaggg gggaatgaaa gaccccacct gtaggtttgg caagctagct 5281 taagtaacgc cattttgcaa ggcatggaaa atacataact gagaatagag aagttcagat 5341 caaggttagg aacagagaga cagcagaata tgggccaaac aggatatctg tggtaagcag 5401 ttcctgcccc ggctcagggc caagaacaga tggtccccag atgcggtccc gccctcagca 5461 gtttctagag aaccatcaga tgtttccagg gtgccccaag gacctgaaat gaccctgtgc 5521 cttatttgaa ctaaccaatc agttcgcttc tcgcttctgt tcgcgcgctt ctgctccccg 5581 agctcaataa aagagcccac aacccctcac tcggcgcgcc agtcctccga tagactgcgt 5641 cgcccgggta cccgtattcc caataaagcc tcttgctgtt tgcatccgaa tcgtggactc 5701 gctgatcctt gggagggtct cctcagattg attgactgcc cacctcgggg gtctttcatt 5761 tggaggttcc accgagattt ggagacccct gcccagggac caccgacccc cccgccggga 5821 ggtaagctgg ccagcggtcg tttcgtgtct gtctctgtct ttgtgcgtgt ttgtgccggc 5881 atctaatgtt tgcgcctgcg tctgtactag ttagctaact agctctgtat ctggcggacc 5941 cgtggtggaa ctgacgagtt ctgaacaccc ggccgcaacc ctgggagacg tcccagggac 6001 tttgggggcc gtttttgtgg cccgacctga ggaagggagt cgatgtggaa tccgaccccg 6061 tcaggatatg tggttctggt aggagacgag aacctaaaac agttcccgcc tccgtctgaa 6121 tttttgcttt cggtttggaa ccgaagccgc gcgtcttgtc tgctgcagcg ctgcagcatc 6181 gttctgtgtt gtctctgtct gactgtgttt ctgtatttgt ctgaaaatta gggccagact 6241 gttaccactc ccttaagttt gaccttaggt cactggaaag atgtcgagcg gatcgctcac 6301 aaccagtcgg tagatgtcaa gaagagacgt tgggttacct tctgctctgc agaatggcca 6361 acctttaacg tcggatggcc gcgagacggc acctttaacc gagacctcat cacccaggtt 6421 aagatcaagg tcttttcacc tggcccgcat ggacacccag accaggtccc ctacatcgtg 6481 acctgggaag ccttggcttt tgacccccct ccctgggtca agccctttgt acaccctaag 6541 cctccgcctc ctcttcctcc atccgccccg tctctccccc ttgaacctcc tcgttcgacc 6601 ccgcctcgat cctcccttta tccagccctc actccttctc taggcgccgg aattcagatc 6661 ttacgtagct agcggatccc aattgcacag ggacagcaga gatccagttt ggttagtacc 6721 gggccctacg cgttactcga gccaaggtcg ggcaggaaga gggcctattt cccatgattc 6781 cttcatattt gcatatacga tacaaggctg ttagagagat aattagaatt aatttgactg 6841 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt 6901 ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt 6961 atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccggg tcttcgagaa 7021 gacgttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa 7081 agtggcaccg agtcggtgct ttttttggat ccaattctac cgggtagggg aggcgctttt 7141 cccaaggcag tctggagcat gcgctttagc agccccgctg ggcacttggc gctacacaag 7201 tggcctctgg cctcgcacac attccacatc caccggtagg cgccaaccgg ctccgttctt 7261 tggtggcccc ttcgcgccac cttctactcc tcccctagtc aggaagttcc cccccgcccc 7321 gcagctcgcg tcgtgcagga cgtgacaaat ggaagtagca cgtctcacta gtctcgtgca 7381 gatggacagc accgctgagc aatggaagcg ggtaggcctt tggggcagcg gccaatagca 7441 gctttgctcc ttcgctttct gggctcagag gctgggaag //